BACKGROUND
archaea are prokaryotes, they resemble bacteria in genome size, genome organization and the absence of a nuclear membrane, but their genetic information storage and expression components are generally more closely related to their eukaryotic than bacterial counterparts  <cit> .
historically, difficulties in manipulating archaea genetically limited archaeal research but, with the discovery that thermococcus kodakarensis is naturally competent for dna uptake and transformation  <cit> , genetic tools have been developed and t. kodakarensis established as a readily tractable experimental model for archaeal and hyperthermophile research  <cit> .
as a fermentative heterotroph that grows rapidly, optimally at 85°c on a range of different substrates, t. kodakarensis offers opportunities to investigate archaeal gene regulation and metabolism under a variety of growth conditions and, as a hydrogen-producer, it has also attracted biotechnology attention.
by using high-throughput rna sequencing , it is now possible to identify essentially all transcripts present in cells  <cit>  and such studies have revealed surprisingly complex transcriptomes in bacteria, with many previously unanticipated non-coding small  and antisense rnas .
to add to this database, and specifically to add to the relatively few rna-seq studies reported to date for archaea, we have used differential rna-seq technology  to identify the transcripts present in t. kodakarensis cells growing on different substrates and in stationary-phase cells.
an automated analysis  <cit>  was used to identify the sites at which the transcripts were initiated  throughout the genome.
based on the conservation of sequences upstream of the tss identified, consensus sequences have been identified for the core elements of t. kodakarensis promoters from which the synthesis of primary, secondary, internal and antisense transcripts is initiated.
RESULTS
t. kodakarensistranscripts and transcription start sites
identifying transcripts and precisely mapping transcription start sites  refines and extends genome annotation and the discovery of regulatory elements that control gene expression.
to obtain this knowledge for t. kodakarensis, we employed differential high-throughput sequencing of cdna libraries  generated from rna preparations isolated from cells grown with and without sulfur, growth conditions that result in different patterns of gene expression and metabolism .
specifically, cdna libraries were generated and sequenced from rna isolated from t. kodakarensis cells growing exponentially  and to stationary phase  in asw-yt medium with sulfur, growing exponentially in asw-yt with pyruvate , and from cells growing exponentially in pyruvate but 20 min after sulfur addition .
the cdnas were generated after first incubating the rna preparations with terminator exonuclease .
tex does not degrade primary transcripts with a 5′-triphosphate  <cit>  but does digest rnas generated by transcript processing that have a 5′-monophosphate.
as a control and to fully document all transcripts, a cdna library  was also generated and sequenced from an aliquot of an rna preparation isolated from the cells growing exponentially with sulfur that was not exposed to tex digestion.
a total of ~ <dig> million cdna sequencing reads were obtained with  <dig>  to  <dig>  million reads generated from each individual library .
after trimming, removal of poly-tailed sequences and sequences shorter than 12 nt, 89% to 98% of the remaining sequences in each library mapped unequivocally to the t. kodakarensis genome  <cit> .
cdna sequences generated from drna-seq libraries cluster at the + <dig> site of the transcript  <cit>  and this enrichment helps identify tss and their associated upstream regulatory motifs.
the sequences obtained were first evaluated, using an automated tss identification method  <cit>  that was provided with the t. kodakarensis genome annotation extended with known and predicted rnas from the rfam database  <cit>  and from the ucsc archaeal genome browser  <cit> .
a total of  <dig>  tss were identified and categorized based on their locations relative to annotated genes .
primary and secondary tss were defined as those initiating transcription on the sense strand and located upstream and within 300 bp of an annotated gene.
the primary tss  was defined as that for which the most sequencing reads were obtained, and all other tss assigned to the same transcriptional unit were designated secondary tss .
tss located within an annotated gene were designated internal tss  and when the tss was located on the antisense strand in, or within 100 bp of an annotated gene, it was designated an antisense tss .
as illustrated , in some cases, an itss was potentially the ptss or stss for a downstream gene and some atss were potentially the ptss or stss for a gene on the complementary dna strand.
in these cases, the tss was included in both categories, generating four categories that in total contained  <dig>  tss .
there are  <dig>  orfs annotated in the t. kodakarensis genome  <cit>  and  <dig>  of these are predicted, by the door <dig> database, to be organized into  <dig> multi-gene operons  <cit> .
a total of  <dig>  ptss were identified, and these include a ptss identified upstream of ~78% of the predicted protein-encoding transcription units.
six tss have been reported from experimental studies of t. kodakarensis, five match the ptss  identified here for that transcriptional unit .
in the sixth case , the ptss identified is 679 bp upstream of the experimentally determined tss  <cit> .
based on an overlapping atga-arrangement of translation initiation and termination codons, tk0670-tk <dig> very likely form an operon with tk <dig> being the promoter-distal gene.
the location of the tss identified here is consistent with the ptss for a transcript of this operon.figure  <dig>
transcription start site  classification.
diagram showing all potential locations of tss.
in this report, they are categorized as primary  or secondary tss  when located ≤300 bp upstream of a gene with transcription on the sense strand of that gene.
they are categorized as internal tss  when located within an annotated gene and antisense tss  when located either in, or within ≤100 bp of a gene, on the antisense strand.
a tss not readily assigned to any of these categories was categorized as an orphan tss .
the distribution and overlap of the tsss identified in each category.
the distribution of lengths of 5′-utrs based on the combined number of ptss and stss that initiate transcription at each position, with  <dig> being the first bp of the translation start codon.
the numbers of tss resulting in 5'-utrs ≤8 nt, and so considered leaderless mrnas, are indicated by the grey bars.
the insert shows the consensus of the rbs identified in t. kodakarensis.
promoters
archaeal promoters typically have a b recognition element  followed by a tata-box and direct transcription initiation ~23 bp downstream from the tata-box sequence  <cit> .
to screen for promoter motifs, the sequences from -50 bp upstream to all identified tss were analyzed using meme <cit> .
this confirmed the presence of a tata-box motif, located at a median distance of 23 bp, upstream of 96% and 88% of all the ptss and stss, respectively .
a bre motif, positioned upstream of the tata-box, at a median distance of 33 bp from the tss, was also clearly conserved although there was more sequence variability in the bres upstream of stss presumably correlating with the stss being so designated based on lower expression.
it is also possible that some tss, designated stss, are not functionally associated with the downstream orf but rather are the tss of orphan transcripts directed by promoters that have more divergent sequences.
bre and tata-box motifs are also present upstream of ~95% of the itss, some of which are likely to be the ptss for downstream genes .
consensus bre and tata-box motifs are also present upstream of most tss that result in the synthesis of orphan small non-coding transcripts  and antisense transcripts  , arguing that these are discrete gene products rather than products of random transcription or rna processing as concluded for the majority of srnas and asrnas in pyrococcus abyssi <cit> .figure  <dig>
consensus promoter motifs.
each frame shows the best ranking sequence motif identified by a meme search  <cit>  performed on a window from -50 bp upstream to the tss for  ptss of annotated orfs,  stss of annotated orfs,  itss and  atss.
an orphan transcript was so designated when there was no detectable association of a tss with an annotated gene, and  shows the consensus promoter motifs for orphan transcripts.
the negative numbers on the x-axis indicate the distance in bp upstream from the identified tss.
5′-untranslated regions  and leaderless mrnas
archaeal mrnas typically have short 5′-utrs and, in some species, many 5′-utrs are ≤8 nt and, as such, are designated as leaderless and are translated using a distinct initiation mechanism .
based on the tss identified, the majority of the mrnas in t. kodakarensis have a 5′-utr between  <dig> and 50 nt in length, with the median length being 16 nt.
a ribosome binding sequence  that conforms to the consensus ggdgrd is present in ~50% of the predicted mrnas .
initially, we identified  <dig> leaderless mrnas based on having a 5′-utr ≤8 nt long, most of which encode proteins with unknown functions although  <dig> have annotated functions related to rna processing and modification.
to confirm that these were leaderless transcripts, a 100 bp window around each tss was checked for the presence of a rbs using fimo <cit>  and for alternative translation initiating codons.
in  <dig> cases, this revealed evidence against the leaderless mrna designation .
most often, a gtg codon was annotated as the translation initiating codon but an atg codon was also present, in-frame, located  <dig> or  <dig> codons downstream within the orf.
with this atg codon designated as the start codon, the 5′-utr was extended and the transcript no longer conformed to the definition of a leaderless mrna.
transcripts with long 5′-untranslated regions
in total,  <dig> mrnas were identified with long 5′-utrs, defined as between  <dig> and 300 nt in length, with a median length of 103 nt.
these 5′-utrs have sequences ranging from 24% to 66% gc, with a mean of 48% gc, only slightly higher than the average 42% gc content of protein-encoding sequences, arguing against secondary structures stabilized by extensive g:c pairing.
most of these mrnas encode hypothetical proteins, but  <dig> encode transporters,  <dig> encode aminoacyl-trna biosynthesis functions and  <dig> encode ribosomal proteins , data consistent with results from p. abyssi and sulfolobus solfataricus <cit> .
transcripts with long 5′-utrs were also identified in methanosarcina mazei, suggesting post-transcription regulatory role  <cit>  but, to date, there is only very limited experimental evidence for archaeal 5′-utrs having regulatory functions  <cit> .
there is strong in silico support for riboswitches  <cit>  and our results confirm the presence of transcripts predicted to function as fluoride-sensing  <cit>  and pre-q <dig> sensing  <cit>  riboswitches in the thermococcales.
the 86 nt putative fluoride-sensing riboswitch, designated the crcb rna, is present in growing cells, but was not detected in rna from stationary phase cells  and, surprisingly, is encoded upstream of tk <dig> rather than tk <dig> , the gene predicted to encode a fluoride exporter  <cit> .
it remains possible that tk0513-tk <dig> are cotranscribed, under some circumstances, but the ptss for tk <dig> is located between tk <dig> and tk <dig> arguing that tk <dig> expression is not predominantly subject to crcb rna regulation .
the presence of the crcb rna in p. abyssi was also confirmed by rna-seq  <cit> .
synthesis of the putative pre-q <dig> sensing riboswitch  <cit> , designated srk <dig> in p. abyssi, is also growth phase regulated in t. kodakarensis but this rna appears to be a small orphan transcript in t. kodakarensis  rather than present within the 5′-utr of a mrna as in p. abyssi <cit> .
although a riboswitch function has not been proven, there is evidence for riboswitch sequences interacting in trans with potential target mrnas and thereby modulating gene expression  <cit> .
the two srk <dig> rnas do have very similar sequences, but they are encoded in different genome contexts.
downstream of the srk <dig> encoding dna in t. kodakarensis  is a bre-tata box sequence and a tss arguing that the immediately downstream gene  is independently transcribed .figure  <dig>
location and expression of potential regulatory rnas.
transcript abundances, based on cdna reads, from regions proposed to function as  a fluoride-sensing riboswitch upstream of tk <dig>  <cit>  and  a pre-q <dig> riboswitch designated srk <dig>   transcripts of ssca  <cit>  present in the 5'-utr of tk <dig>   transcripts of trnalys  present within the 5'-utr of tk <dig>  an antisense rna is also transcribed from the tk <dig> region.
the numbers of cdna reads from transcripts present in t. kodakarensis cells growing exponentially with sulfur  and in stationary phase in sulfur medium , growing exponentially in pyruvate medium before  and 20 min after sulfur addition  are given by the peak heights.
data from the control library  not digested with tex are shown in grey.
the relative abundance scales on the right of each panel allow direct comparisons of all data in that panel.
the black scale bar in the top right corner of each panel is corresponds to 100 nt.
klein et al.
<cit>  predicted the presence of a regulatory rna, designated the ssca rna, located upstream of the gene  that encodes the translation elongation factor 1α .
our results confirm that the ssca rna is present and abundant in growing t. kodakarensis cells and that it is located within the 118 nt 5′-utr of the tk <dig> mrna .
the function of the ssca rna remains to be determined, but its abundance increases with sulfur addition, and its location suggests a role in regulating translation.
the transcript of a nearby gene , that encodes a dead-box rna helicase, was also reported to have a long 158 nt 5′-utr  <cit> .
our results confirm the presence of this long 5′-utr, demonstrate that it is actually 159 nt and reveal that it contains a trnalys  apparently therefore co-transcribed with tk <dig> .
a short  antisense rna is also transcribed from within the tk <dig> that is less abundant in cells growing with sulfur.
internal transcription start sites
in total,  <dig> tss were identified within protein-encoding sequences and so are designated itss.
of these,  <dig>  are close to the 3′-terminus of an orf and  <dig>  were automatically assigned by the analysis software as the ptss of a downstream gene.
a detailed individual review confirmed that  <dig> were most likely the ptss of a downstream gene with bre-tata box motifs located appropriately upstream within the orf.
as illustrated , these include the tss for tk <dig> and tk <dig>  the genes that encode the rpol and rpon subunits of the t. kodakarensis rna polymerase, respectively.
there is also a region near the 3′-terminus of the rpol transcript that resists tex digestion, consistent with the sequence folding into a stable base-paired secondary structure.
this is not a feature unique to the rpol transcript.
additional file 6: figure s <dig> shows several more examples of 3′-terminal regions of t. kodakarensis transcripts that resist tex digestion and could fold into stable base-paired secondary structures.figure  <dig>
internal transcription start sites.
the genome organizations surrounding  rpol  and  rpon .
the promoter motifs for the ptss of tk <dig> and tkt <dig> and for the itss identified for rpol and rpon are shown below the panels.
the abundances of transcripts present in t. kodakarensis cells growing exponentially  and in stationary phase  in sulfur medium, growing exponentially in pyruvate medium before  and 20 min after sulfur addition  are given by the peak heights.
data from the control library  not digested with tex are shown in grey.
the position of the sequence near the 3′-terminus of rpol that, when transcribed, is predicted to form a stable rna hairpin structure is indicated .
the black scale bar in the top right corner of each panel is corresponds to 100 nt.
initially,  <dig> itss were identified near the 5′-termini of orfs but, in  <dig> cases, translation initiation could occur at an alternative start codon located in-frame but farther downstream, which would place these tss within the upstream intergenic region .
as an example, tk <dig> is annotated as encoding a mini-chromosome maintenance protein  with an atypically long n-terminal extension  <cit> .
in the drna-seq data, an itss is present within tk <dig> and transcripts initiated at this site would have a rbs and encode a standard mcm with no extension .
interestingly, tk <dig> that also encodes a mcm homologue , also contains an itss positioned appropriately downstream of a bre-tata-box sequence and upstream of a potential rbs and an in-frame gtg start codon, although translation initiated at this gtg would result in a much truncated mcm <dig>  tk <dig> is also apparently the promoter distal gene in a tk1619-tk <dig> operon for which a well-defined ptss was identified .
the mcm <dig> protein is synthesized initially as a precursor, containing a homing-endonuclease intein and as the itss, its putative promoter and the downstream gtg codon are all within the intein-encoding region, these regulatory elements and/or the truncated mcm <dig> may participate in mcm <dig> maturation .figure  <dig>
transcription of tk <dig>  and tk <dig> .
as annotated in the t. kodakarensis genome  <cit> , tk <dig> has an atypical 5'-extension, here shown by broken lines.
the location of the tss identified for tk <dig> and the sequence downstream that could function as a translation start site are shown.
with the tss re-categorized from itss to ptss and translation initiated at the boxed gtg codon, the mcm <dig> generated has a standard mcm structure.
the organization of the tk1619-tk <dig>  region.
the locations and upstream promoter sequences for the ptss and an itss within tk <dig> are indicated.
a putative gtg translation initiation codon downstream of the itss is boxed.
in both panels the protein domains, identified in the ncbi conserved domain database  <cit> , are shown in dark grey.
the abundances of transcripts present in t. kodakarensis cells growing exponentially  and in stationary phase  in sulfur medium, growing exponentially in pyruvate medium before  and 20 min after sulfur addition  are given by the peak heights.
data from the control library  not digested with tex are shown in grey.
the relative abundance scales on the right of each panel allow direct comparisons of all data in that panel.
the black scale bar in the top right corner of each panel is corresponds to 100 nt.
for all but three of the remaining itss, an appropriately positioned bre-tata box sequence is readily apparent consistent with the downstream region of the orf being transcribed from two structurally separate promoters.
in some, but not all cases, there are also sequences that could function as a rbs and translation initiating codon, within the orf, downstream of the itss.
in the absence of such translation initiating elements, transcription from the itss presumably results in a non-coding rna as exemplified by the hgcc transcripts.
hgcc rnas were originally identified by bioinformatically and their expression then verified by northern blot analysis  <cit> .
as documented in the rfam database, homologous sequences are found in  <dig> archaea, most are hyperthermophiles, but some are halophiles  <cit> .
the t. kodakarensis genome has sequences encoding seven hgcc transcripts  <cit>  and the drna-seq data confirm that five of these, designated tk hgcc <dig> to hgcc <dig>  are present in t. kodakarensis cells .
as reported for an hgcc in p. furiosus <cit> , tk hgcc <dig>  tk hgcc <dig> and tk hgcc <dig> are transcribed from transposase encoding sequences.
the p. furiosus hgcc transcript is, however, an antisense transcript relative to the transposase gene whereas tk hgcc <dig>  hgcc <dig> and hgcc <dig> are transcribed in the same direction from itsss within the transposase genes, tk <dig>  tk <dig> and tk <dig>  respectively .
tk hgcc <dig> and tk hgcc <dig> are not associated with transposase genes.
hgcc <dig> has an itss within tk <dig> , and the tss of hgcc <dig> is also the ptss of tk <dig> as hgcc <dig> is encoded within the 5′-region of tk <dig>  .
an antisense rna, complementary to part of hgcc <dig>  is also transcribed from the tk <dig> region that could function in trans as a regulator of hgcc synthesis and/or function .
antisense transcripts in t. kodakarensis
before the advent of deep sequencing, most non-coding rnas were identified through sequence conservation and predictions for conserved rna folding, with screening for non-coding rnas generally avoiding orfs and their complementary antisense regions.
however, as transcriptome data have accumulated, it has become increasingly clear that antisense transcription is a major feature of prokaryotic and eukaryotic genome expression  <cit> .
the drna-seq data identified  <dig>  atss, sites at which antisense transcription is initiated on the t. kodakarensis genome.
the atss are not evenly distributed.
inspection of 150 bp windows around translation start and stop codons revealed that 58% of the atss are located near gene termini with  <dig> and  <dig> antisense transcripts overlapping the 5′- and 3′-terminus, respectively, of an orf.
a similar enrichment of antisense transcription across gene termini has also been observed in other archaea, with antisense transcripts often associated with transposase-encoding genes .
an atss is associated with all seven genes  annotated as encoding transposases in the t. kodakarensis genome  <cit> .
to identify any additional preferential associations of atss with specific functions, the atss locations were evaluated relative to the pathways and functions defined in the kegg database  <cit> .
antisense transcripts are associated with  <dig>   <dig>   <dig> and  <dig> orfs that encode proteins involved in amino acid, purine, pyrimidine and central carbon metabolism, respectively,  <dig> that participate in ribosome biogenesis and  <dig> that encode abc transporters.
at least some of these antisense rnas most likely interact with the complementary sense mrna but, as yet, there is no direct experimental evidence for such an interaction in vivo.
this has been documented experimentally in vitro for an antisense rna from m. mazei that sequestered the rbs of the target mrna  <cit> .
known or predicted small non-coding rnas
the rfam and ucsc databases predict that ~ <dig> small non-coding rnas are encoded in the t. kodakarensis genome  <cit> .
most are classified as snornas, archaeal counterparts of eukaryotic snornas  that direct 2′-o-methylation  and pseudouridylation  of transfer  and ribosomal rnas .
we verified the presence of  <dig> of these small non-coding rnas ;  <dig> designated as c/d box and  <dig> as h/aca box snornas.
of these,  <dig> are not recognizably linked to annotated genes and so are classified as orphan snornas.
there are  <dig> potential snornas encoded within 5′-utrs and  <dig> within the 5′-coding region of orfs that, presumably, must be released by transcript processing.
tko-sr <dig>  for example, appears to be co-transcribed with a trnaarg and is then likely released from the co-transcript by rnase z cleavage, as observed in nanoarchaeum equitans and in some plants  <cit> .
in some cases, the presence of a k-turn motif near the 5′-terminus, the region predicted to be a snorna may, in fact, be a cis-regulatory element rather than a snorna.
k-turn motifs are important structural elements in riboswitches  <cit> .
alternatively, as sequences that conform to ribosomal protein l7ae  binding sites are present in several of these transcripts, including those encoding the anop <dig>  and cbf5p  components of the snorna guide complexes, these transcripts might be processed by complexes containing l7ae and possibly rnase p  <cit> .
seventeen of the potential snornas are antisense transcripts,  <dig> of which are transcribed from dna that includes the 3′-terminus of the complementary sense gene.
in  <dig> cases, a snorna is transcribed convergent to an antisense rna, a gene organization also documented in s. solfataricus, n. equitans and pyrobaculum species  <cit> .
deep sequencing identified  <dig> sense and  <dig> antisense non-coding rnas in p. abyssi ge <dig>  <cit> .
based on a blast search,  <dig> of these are also encoded in the t. kodakarensis genome, of which  <dig> are clearly represented in the drna-seq libraries.
a further  <dig> orphan small non-coding rnas predicted and/or documented to be present in pyrococcus species  <cit>   and the small non-coding rnas, designated crispr rnas , predicted to be transcribed from the three crispr loci in the t. kodakarensis genome are also present in the t. kodakarensis drna-seq libraries  <cit> .
transcription of a crispr locus generates a long transcript that is cleaved, first releasing immature crrnas with 8 nt 5′-extensions and variable 3′-termini.
these are then trimmed to produce the mature crrnas.
the tss identified are fully consistent with the locations predicted for promoters within the crispr loci and with this transcript processing  <cit> .
orphan small non-coding rnas
in addition to the  <dig> orphan snornas detected and the non-coding small rnas also identified in pyrococcus species , the drna-seq libraries document the presence of  <dig> previously unrecognized small transcripts, including an unrecognized snorna .
orfs > <dig> codons are not present in any of these transcripts and only  <dig>  are conserved in other archaea  arguing for t. kodakarensis specific functions.
historically, computer screening for non-coding rnas in hyperthermophiles has employed a high gc content  as a filter  <cit>  but only 30% of the non-coding rnas now identified in t. kodakarensis have gc contents >50% and only  <dig> of those first reported here meet this criterion.
given that the sequences of these transcripts are, on average ~43% gc, they seem unlikely to have stable base-paired secondary structures in vitro at the 85°c optimum growth temperature of t. kodakarensis but may have secondary structures stabilized in vivo by protein interactions.
this is predicted for lsm binding to several srnas in h. volcanii <cit>  and the t. kodakarensis genome encodes several rna-binding proteins  <cit> , in addition to lsm, with alba  and a putative rna-binding protein encoded by tk <dig> likely present in very high abundance .table  <dig>
protein-encoding genes with the highest numbers of reads per orf in the pyruvate  and sulfur  cdna libraries
1
exp
exp
1the tkxxxx numerical gene designation for the promoter proximal gene in the transcription units that had ≥ <dig>  reads .
the genes are listed in decreasing order of cdna reads, with shading identifying genes present in both lists.
2the automated analysis assigned the cdna reads to genes tk <dig>  tk <dig> and tk <dig>  manual inspection revealed the presence of tko-sr <dig>  tko-sr <dig> and tko-sr <dig>  respectively, within the 5′-terminal region of each transcript.
trnas, rrnas, rnase p and 7s rnas
all of the trnas and rrnas annotated in the t. kodakarensis genome are present and fully covered in the drna-seq libraries .
there are also antisense transcripts present complementary to six trnas and one trnathr is, itself, an antisense transcript of tk <dig> .
as described above, one trnaarg appears to be cotranscribed with a snorna  and a snorna  encoding sequence is located immediately upstream, and is likely within the 5′-leader region and so cotranscribed with the 16s rrna-trnaala-23s rrna operon
the drna-seq libraries also confirm the presence of the rnase p rna and 7s  rna in t. kodakarensis cells although the 7s rna is transcribed from the dna strand opposite to that stated in the genome annotation  <cit> .
the drna-seq data are convincing  and in agreement with the rfam database  <cit> .
growth and media-dependent transcription
based on previous studies of p. furiosus <cit>  and t. kodakarensis <cit> , we generated cdna libraries from cells growing in media with sulfur  or pyruvate  to increase the number of tss likely identified.
we also generated cdna libraries from cells grown with sulfur to stationary phase  and from cells growing with pyruvate but with sulfur added 20 min before rna isolation .
the rna preparations were subjected to tex digestion before cdna synthesis and, given in vivo transcript processing and in vitro fragmentation during purification, the resulting cdna libraries were, as expected, enriched for 5′-terminal sequences.
this facilitates the identification of tss but, assuming that the number of cdna reads correlates with transcript abundance, the drna-seq data also provide a semi-quantitative overview of global genome expression and are consistent with previous observations of substrate-dependent specific gene expression.
based on the number of cdna reads, there is little or no transcription from ~35% of transcriptional units  in cells growing in pyruvate medium, of ~28% of the tus in cells growing in sulfur medium, and of ~87% of tus in stationary phase cells.
transcript abundances vary substantially, but < <dig> % are present >1000-fold above the minimal detectable level .
consistent with constitutively high expression and/or transcript stability,  <dig> of the  <dig> most abundant transcripts were the same in rna preparations from cells growing in sulfur or pyruvate medium
schut et al  <cit>  documented that when sulfur was added to p. furiosus cultures growing in pyruvate medium, h2s replaced h <dig> as an end-product of metabolism and transcription of the mbh operon was replaced by transcription of the mbx operon.
this metabolic shift also occurs in t. kodakarensis <cit>  and, based on the numbers of cdna reads obtained from rna preparations isolated from t. kodakarensis cells growing in pyruvate medium before and after sulfur addition, the transcription regulation is also conserved in t. kodakarensis.
as in p. furiosus <cit> , transcription of the mbh operon  that encodes the h2-generating hydrogenase is rapidly and almost fully terminated following sulfur addition and replaced by transcription of the mbx operon  that encodes the h2s-generating enzyme.
.figure  <dig>
heatmap comparison of changes in gene expression after sulfur addition.
changes in transcript abundance are shown, on a log2-fold scale, for the tk genes listed.
comparisons were made of the cdna libraries generated from rna isolated before and 20 min after addition of sulfur to t. kodakarensis cells growing exponentially in pyruvate.
values were calculated based on changes in the abundance of cdna reads, as described in materials and methods.
the t. kodakarensis data are aligned and compared with the microarray hybridization results reported for sulfur-induced changes in transcription of the homologous mbx and mbh operons, and related homologous genes, in p. furiosus <cit> .
the reductant needed to generate h <dig> and/or h2s is most likely supplied by a reduced ferredoxin  <cit>  but there are three candidate ferredoxins encoded in the t. kodakarensis genome  <cit> .
based on cdna reads, transcripts of tk <dig>  are very abundant under all of the growth conditions investigated , indicative of ferredoxin- <dig> participating in many metabolic pathways, although there is a ~2-fold decrease after sulfur addition to pyruvate growing cells.
ferredoxin- <dig> is encoded by tk <dig>  the middle gene in a three gene operon  that also encodes surr , a redox-responsive transcription regulator of many genes involved in sulfur metabolism  <cit> .
the extent of ferredoxin- <dig> reduction could provide redox-state information to surr, and so modulate surr activity, but sulfur addition had little effect on the abundance of tk1086-tk <dig> transcripts .
in contrast, there was a ~6-fold decrease in tk <dig> transcripts  following sulfur addition to pyruvate growing cells, arguing that ferredoxin- <dig> is likely the predominant electron donor for h <dig> production by the mbh encoded hydrogenase .
intriguingly, an antisense transcript is also generated from the tk <dig> region that has increased expression in the presence of sulfur , and a surr binding site overlaps the tata box of the promoter for this antisense transcript.
the antisense rna is therefore likely part of the surr regulon, and tk <dig> expression and so ferredoxin- <dig> synthesis may be indirectly subject to surr regulation through post-transcription regulation by the antisense rna.
discussion and
CONCLUSIONS
the results obtained establish that transcription initiation occurs at > <dig>  sites around the t. kodakarensis genome  and recognizable bre-tata-box promoter elements are appropriately located upstream of ~78% of all the t. kodakarensis transcription units identified.
as reported for other archaea <cit> , many promoters and tss are embedded within orfs, and antisense transcription occurs extensively throughout the t. kodakarensis genome adding significantly to the genome complexity, and predicting a major involvement of antisense transcripts in regulating gene expression.
t. kodakarensis cells contain many small non-coding rnas, some previously identified or predicted including two candidate riboswitches  <cit>  but also many previously unanticipated rnas .
as in p. abyssi <cit> , some have relatively au-rich sequences, in contrast to the high gc content of t. kodakarensis trnas and rrnas, and contrary to the expectation  <cit>  that small non-coding rnas in hyperthermophiles would be gc-rich to stabilize secondary structures.
the sequences of most of these small au-rich rnas do not readily fold into canonical base-paired secondary structures, and may function as unstructured molecules  <cit> , but could have secondary and tertiary structures stabilized in vivo by nucleic acid and protein interactions  <cit> .
most 5′-utrs in t. kodakarensis are short and some are leaderless .
but, as in other archaea <cit> , there are also mrnas with long 5′-utrs that are consistent and predictive of cis-regulatory elements, although there is no direct experimental support to date for regulation in vivo by attenuators or riboswitches in archaea.
the most exciting and experimentally-challenging conclusion from this, and from all other archaeal transcriptome studies to date, is that archaeal cells contain many different, often abundant and apparently non-coding small rnas.
t. kodakarensis appears typical; it has a very small genome  tightly packed with orfs but also with genes that encode non-translated rnas and so likely has widespread rna-based regulation.
historically, rna secondary structure was sought and equated with non-coding rna function but it is now clear that such structure is not mandatory.
for example eukaryotic sirnas and lncrnas apparently bind their rna and protein targets, and carry out their regulatory functions, without extensive structure  <cit> .
given that gene expression in archaea, and particularly transcription-related features are simpler but have many features in common with their eukaryotic counterparts, it seems likely that investigating rna-based regulation in archaea, with t. kodakarensis providing a model system  <cit> , will generate results that are valuable and legitimately extrapolated into eukaryotic gene expression.

 archaea are prokaryotes, they resemble bacteria in genome size, genome organization and the absence of a nuclear membrane, but their genetic information storage and expression components are generally more closely related to their eukaryotic than bacterial counterparts. historically, difficulties in manipulating archaea genetically limited archaeal research but, with the discovery that thermococcus kodakarensis is naturally competent for dna uptake and transformation, genetic tools have been developed and t. kodakarensis established as a readily tractable experimental model for archaeal and hyperthermophile research. as a fermentative heterotroph that grows rapidly, optimally at 85°c on a range of different substrates, t. kodakarensis offers opportunities to investigate archaeal gene regulation and metabolism under a variety of growth conditions and, as a hydrogen-producer, it has also attracted biotechnology attention. by using high-throughput rna sequencing, it is now possible to identify essentially all transcripts present in cells and such studies have revealed surprisingly complex transcriptomes in bacteria, with many previously unanticipated non-coding small and antisense rnas. to add to this database, and specifically to add to the relatively few rna-seq studies reported to date for archaea, we have used differential rna-seq technology to identify the transcripts present in t. kodakarensis cells growing on different substrates and in stationary-phase cells. an automated analysis was used to identify the sites at which the transcripts were initiated throughout the genome. based on the conservation of sequences upstream of the tss identified, consensus sequences have been identified for the core elements of t. kodakarensis promoters from which the synthesis of primary, secondary, internal and antisense transcripts is initiated.  t. kodakarensistranscripts and transcription start sites identifying transcripts and precisely mapping transcription start sites refines and extends genome annotation and the discovery of regulatory elements that control gene expression. to obtain this knowledge for t. kodakarensis, we employed differential high-throughput sequencing of cdna libraries generated from rna preparations isolated from cells grown with and without sulfur, growth conditions that result in different patterns of gene expression and metabolism. specifically, cdna libraries were generated and sequenced from rna isolated from t. kodakarensis cells growing exponentially and to stationary phase in asw-yt medium with sulfur, growing exponentially in asw-yt with pyruvate, and from cells growing exponentially in pyruvate but 20 min after sulfur addition. the cdnas were generated after first incubating the rna preparations with terminator exonuclease. tex does not degrade primary transcripts with a 5′-triphosphate but does digest rnas generated by transcript processing that have a 5′-monophosphate. as a control and to fully document all transcripts, a cdna library was also generated and sequenced from an aliquot of an rna preparation isolated from the cells growing exponentially with sulfur that was not exposed to tex digestion. a total of ~ million cdna sequencing reads were obtained with to million reads generated from each individual library. after trimming, removal of poly-tailed sequences and sequences shorter than 12 nt, 89% to 98% of the remaining sequences in each library mapped unequivocally to the t. kodakarensis genome. cdna sequences generated from drna-seq libraries cluster at the + site of the transcript and this enrichment helps identify tss and their associated upstream regulatory motifs. the sequences obtained were first evaluated, using an automated tss identification method that was provided with the t. kodakarensis genome annotation extended with known and predicted rnas from the rfam database and from the ucsc archaeal genome browser. a total of tss were identified and categorized based on their locations relative to annotated genes. primary and secondary tss were defined as those initiating transcription on the sense strand and located upstream and within 300 bp of an annotated gene. the primary tss was defined as that for which the most sequencing reads were obtained, and all other tss assigned to the same transcriptional unit were designated secondary tss. tss located within an annotated gene were designated internal tss and when the tss was located on the antisense strand in, or within 100 bp of an annotated gene, it was designated an antisense tss. as illustrated, in some cases, an itss was potentially the ptss or stss for a downstream gene and some atss were potentially the ptss or stss for a gene on the complementary dna strand. in these cases, the tss was included in both categories, generating four categories that in total contained tss. there are orfs annotated in the t. kodakarensis genome and of these are predicted, by the door database, to be organized into multi-gene operons. a total of ptss were identified, and these include a ptss identified upstream of ~78% of the predicted protein-encoding transcription units. six tss have been reported from experimental studies of t. kodakarensis, five match the ptss identified here for that transcriptional unit. in the sixth case, the ptss identified is 679 bp upstream of the experimentally determined tss. based on an overlapping atga-arrangement of translation initiation and termination codons, tk0670-tk very likely form an operon with tk being the promoter-distal gene. the location of the tss identified here is consistent with the ptss for a transcript of this operon.figure transcription start site classification. diagram showing all potential locations of tss. in this report, they are categorized as primary or secondary tss when located ≤300 bp upstream of a gene with transcription on the sense strand of that gene. they are categorized as internal tss when located within an annotated gene and antisense tss when located either in, or within ≤100 bp of a gene, on the antisense strand. a tss not readily assigned to any of these categories was categorized as an orphan tss. the distribution and overlap of the tsss identified in each category. the distribution of lengths of 5′-utrs based on the combined number of ptss and stss that initiate transcription at each position, with being the first bp of the translation start codon. the numbers of tss resulting in 5'-utrs ≤8 nt, and so considered leaderless mrnas, are indicated by the grey bars. the insert shows the consensus of the rbs identified in t. kodakarensis. promoters archaeal promoters typically have a b recognition element followed by a tata-box and direct transcription initiation ~23 bp downstream from the tata-box sequence. to screen for promoter motifs, the sequences from -50 bp upstream to all identified tss were analyzed using meme. this confirmed the presence of a tata-box motif, located at a median distance of 23 bp, upstream of 96% and 88% of all the ptss and stss, respectively. a bre motif, positioned upstream of the tata-box, at a median distance of 33 bp from the tss, was also clearly conserved although there was more sequence variability in the bres upstream of stss presumably correlating with the stss being so designated based on lower expression. it is also possible that some tss, designated stss, are not functionally associated with the downstream orf but rather are the tss of orphan transcripts directed by promoters that have more divergent sequences. bre and tata-box motifs are also present upstream of ~95% of the itss, some of which are likely to be the ptss for downstream genes. consensus bre and tata-box motifs are also present upstream of most tss that result in the synthesis of orphan small non-coding transcripts and antisense transcripts, arguing that these are discrete gene products rather than products of random transcription or rna processing as concluded for the majority of srnas and asrnas in pyrococcus abyssi.figure consensus promoter motifs. each frame shows the best ranking sequence motif identified by a meme search performed on a window from -50 bp upstream to the tss for ptss of annotated orfs, stss of annotated orfs, itss and atss. an orphan transcript was so designated when there was no detectable association of a tss with an annotated gene, and shows the consensus promoter motifs for orphan transcripts. the negative numbers on the x-axis indicate the distance in bp upstream from the identified tss. 5′-untranslated regions and leaderless mrnas archaeal mrnas typically have short 5′-utrs and, in some species, many 5′-utrs are ≤8 nt and, as such, are designated as leaderless and are translated using a distinct initiation mechanism. based on the tss identified, the majority of the mrnas in t. kodakarensis have a 5′-utr between and 50 nt in length, with the median length being 16 nt. a ribosome binding sequence that conforms to the consensus ggdgrd is present in ~50% of the predicted mrnas. initially, we identified leaderless mrnas based on having a 5′-utr ≤8 nt long, most of which encode proteins with unknown functions although have annotated functions related to rna processing and modification. to confirm that these were leaderless transcripts, a 100 bp window around each tss was checked for the presence of a rbs using fimo and for alternative translation initiating codons. in cases, this revealed evidence against the leaderless mrna designation. most often, a gtg codon was annotated as the translation initiating codon but an atg codon was also present, in-frame, located or codons downstream within the orf. with this atg codon designated as the start codon, the 5′-utr was extended and the transcript no longer conformed to the definition of a leaderless mrna. transcripts with long 5′-untranslated regions in total, mrnas were identified with long 5′-utrs, defined as between and 300 nt in length, with a median length of 103 nt. these 5′-utrs have sequences ranging from 24% to 66% gc, with a mean of 48% gc, only slightly higher than the average 42% gc content of protein-encoding sequences, arguing against secondary structures stabilized by extensive g:c pairing. most of these mrnas encode hypothetical proteins, but encode transporters, encode aminoacyl-trna biosynthesis functions and encode ribosomal proteins, data consistent with results from p. abyssi and sulfolobus solfataricus. transcripts with long 5′-utrs were also identified in methanosarcina mazei, suggesting post-transcription regulatory role but, to date, there is only very limited experimental evidence for archaeal 5′-utrs having regulatory functions. there is strong in silico support for riboswitches and our results confirm the presence of transcripts predicted to function as fluoride-sensing and pre-q sensing riboswitches in the thermococcales. the 86 nt putative fluoride-sensing riboswitch, designated the crcb rna, is present in growing cells, but was not detected in rna from stationary phase cells and, surprisingly, is encoded upstream of tk rather than tk, the gene predicted to encode a fluoride exporter. it remains possible that tk0513-tk are cotranscribed, under some circumstances, but the ptss for tk is located between tk and tk arguing that tk expression is not predominantly subject to crcb rna regulation. the presence of the crcb rna in p. abyssi was also confirmed by rna-seq. synthesis of the putative pre-q sensing riboswitch, designated srk in p. abyssi, is also growth phase regulated in t. kodakarensis but this rna appears to be a small orphan transcript in t. kodakarensis rather than present within the 5′-utr of a mrna as in p. abyssi. although a riboswitch function has not been proven, there is evidence for riboswitch sequences interacting in trans with potential target mrnas and thereby modulating gene expression. the two srk rnas do have very similar sequences, but they are encoded in different genome contexts. downstream of the srk encoding dna in t. kodakarensis is a bre-tata box sequence and a tss arguing that the immediately downstream gene is independently transcribed.figure location and expression of potential regulatory rnas. transcript abundances, based on cdna reads, from regions proposed to function as a fluoride-sensing riboswitch upstream of tk and a pre-q riboswitch designated srk transcripts of ssca present in the 5'-utr of tk transcripts of trnalys present within the 5'-utr of tk an antisense rna is also transcribed from the tk region. the numbers of cdna reads from transcripts present in t. kodakarensis cells growing exponentially with sulfur and in stationary phase in sulfur medium, growing exponentially in pyruvate medium before and 20 min after sulfur addition are given by the peak heights. data from the control library not digested with tex are shown in grey. the relative abundance scales on the right of each panel allow direct comparisons of all data in that panel. the black scale bar in the top right corner of each panel is corresponds to 100 nt. klein et al. predicted the presence of a regulatory rna, designated the ssca rna, located upstream of the gene that encodes the translation elongation factor 1α. our results confirm that the ssca rna is present and abundant in growing t. kodakarensis cells and that it is located within the 118 nt 5′-utr of the tk mrna. the function of the ssca rna remains to be determined, but its abundance increases with sulfur addition, and its location suggests a role in regulating translation. the transcript of a nearby gene, that encodes a dead-box rna helicase, was also reported to have a long 158 nt 5′-utr. our results confirm the presence of this long 5′-utr, demonstrate that it is actually 159 nt and reveal that it contains a trnalys apparently therefore co-transcribed with tk. a short antisense rna is also transcribed from within the tk that is less abundant in cells growing with sulfur. internal transcription start sites in total, tss were identified within protein-encoding sequences and so are designated itss. of these, are close to the 3′-terminus of an orf and were automatically assigned by the analysis software as the ptss of a downstream gene. a detailed individual review confirmed that were most likely the ptss of a downstream gene with bre-tata box motifs located appropriately upstream within the orf. as illustrated, these include the tss for tk and tk the genes that encode the rpol and rpon subunits of the t. kodakarensis rna polymerase, respectively. there is also a region near the 3′-terminus of the rpol transcript that resists tex digestion, consistent with the sequence folding into a stable base-paired secondary structure. this is not a feature unique to the rpol transcript. additional file 6: figure s shows several more examples of 3′-terminal regions of t. kodakarensis transcripts that resist tex digestion and could fold into stable base-paired secondary structures.figure internal transcription start sites. the genome organizations surrounding rpol and rpon. the promoter motifs for the ptss of tk and tkt and for the itss identified for rpol and rpon are shown below the panels. the abundances of transcripts present in t. kodakarensis cells growing exponentially and in stationary phase in sulfur medium, growing exponentially in pyruvate medium before and 20 min after sulfur addition are given by the peak heights. data from the control library not digested with tex are shown in grey. the position of the sequence near the 3′-terminus of rpol that, when transcribed, is predicted to form a stable rna hairpin structure is indicated. the black scale bar in the top right corner of each panel is corresponds to 100 nt. initially, itss were identified near the 5′-termini of orfs but, in cases, translation initiation could occur at an alternative start codon located in-frame but farther downstream, which would place these tss within the upstream intergenic region. as an example, tk is annotated as encoding a mini-chromosome maintenance protein with an atypically long n-terminal extension. in the drna-seq data, an itss is present within tk and transcripts initiated at this site would have a rbs and encode a standard mcm with no extension. interestingly, tk that also encodes a mcm homologue, also contains an itss positioned appropriately downstream of a bre-tata-box sequence and upstream of a potential rbs and an in-frame gtg start codon, although translation initiated at this gtg would result in a much truncated mcm tk is also apparently the promoter distal gene in a tk1619-tk operon for which a well-defined ptss was identified. the mcm protein is synthesized initially as a precursor, containing a homing-endonuclease intein and as the itss, its putative promoter and the downstream gtg codon are all within the intein-encoding region, these regulatory elements and/or the truncated mcm may participate in mcm maturation.figure transcription of tk and tk. as annotated in the t. kodakarensis genome, tk has an atypical 5'-extension, here shown by broken lines. the location of the tss identified for tk and the sequence downstream that could function as a translation start site are shown. with the tss re-categorized from itss to ptss and translation initiated at the boxed gtg codon, the mcm generated has a standard mcm structure. the organization of the tk1619-tk region. the locations and upstream promoter sequences for the ptss and an itss within tk are indicated. a putative gtg translation initiation codon downstream of the itss is boxed. in both panels the protein domains, identified in the ncbi conserved domain database, are shown in dark grey. the abundances of transcripts present in t. kodakarensis cells growing exponentially and in stationary phase in sulfur medium, growing exponentially in pyruvate medium before and 20 min after sulfur addition are given by the peak heights. data from the control library not digested with tex are shown in grey. the relative abundance scales on the right of each panel allow direct comparisons of all data in that panel. the black scale bar in the top right corner of each panel is corresponds to 100 nt. for all but three of the remaining itss, an appropriately positioned bre-tata box sequence is readily apparent consistent with the downstream region of the orf being transcribed from two structurally separate promoters. in some, but not all cases, there are also sequences that could function as a rbs and translation initiating codon, within the orf, downstream of the itss. in the absence of such translation initiating elements, transcription from the itss presumably results in a non-coding rna as exemplified by the hgcc transcripts. hgcc rnas were originally identified by bioinformatically and their expression then verified by northern blot analysis. as documented in the rfam database, homologous sequences are found in archaea, most are hyperthermophiles, but some are halophiles. the t. kodakarensis genome has sequences encoding seven hgcc transcripts and the drna-seq data confirm that five of these, designated tk hgcc to hgcc are present in t. kodakarensis cells. as reported for an hgcc in p. furiosus, tk hgcc tk hgcc and tk hgcc are transcribed from transposase encoding sequences. the p. furiosus hgcc transcript is, however, an antisense transcript relative to the transposase gene whereas tk hgcc hgcc and hgcc are transcribed in the same direction from itsss within the transposase genes, tk tk and tk respectively. tk hgcc and tk hgcc are not associated with transposase genes. hgcc has an itss within tk, and the tss of hgcc is also the ptss of tk as hgcc is encoded within the 5′-region of tk. an antisense rna, complementary to part of hgcc is also transcribed from the tk region that could function in trans as a regulator of hgcc synthesis and/or function. antisense transcripts in t. kodakarensis before the advent of deep sequencing, most non-coding rnas were identified through sequence conservation and predictions for conserved rna folding, with screening for non-coding rnas generally avoiding orfs and their complementary antisense regions. however, as transcriptome data have accumulated, it has become increasingly clear that antisense transcription is a major feature of prokaryotic and eukaryotic genome expression. the drna-seq data identified atss, sites at which antisense transcription is initiated on the t. kodakarensis genome. the atss are not evenly distributed. inspection of 150 bp windows around translation start and stop codons revealed that 58% of the atss are located near gene termini with and antisense transcripts overlapping the 5′- and 3′-terminus, respectively, of an orf. a similar enrichment of antisense transcription across gene termini has also been observed in other archaea, with antisense transcripts often associated with transposase-encoding genes. an atss is associated with all seven genes annotated as encoding transposases in the t. kodakarensis genome. to identify any additional preferential associations of atss with specific functions, the atss locations were evaluated relative to the pathways and functions defined in the kegg database. antisense transcripts are associated with and orfs that encode proteins involved in amino acid, purine, pyrimidine and central carbon metabolism, respectively, that participate in ribosome biogenesis and that encode abc transporters. at least some of these antisense rnas most likely interact with the complementary sense mrna but, as yet, there is no direct experimental evidence for such an interaction in vivo. this has been documented experimentally in vitro for an antisense rna from m. mazei that sequestered the rbs of the target mrna. known or predicted small non-coding rnas the rfam and ucsc databases predict that ~ small non-coding rnas are encoded in the t. kodakarensis genome. most are classified as snornas, archaeal counterparts of eukaryotic snornas that direct 2′-o-methylation and pseudouridylation of transfer and ribosomal rnas. we verified the presence of of these small non-coding rnas; designated as c/d box and as h/aca box snornas. of these, are not recognizably linked to annotated genes and so are classified as orphan snornas. there are potential snornas encoded within 5′-utrs and within the 5′-coding region of orfs that, presumably, must be released by transcript processing. tko-sr for example, appears to be co-transcribed with a trnaarg and is then likely released from the co-transcript by rnase z cleavage, as observed in nanoarchaeum equitans and in some plants. in some cases, the presence of a k-turn motif near the 5′-terminus, the region predicted to be a snorna may, in fact, be a cis-regulatory element rather than a snorna. k-turn motifs are important structural elements in riboswitches. alternatively, as sequences that conform to ribosomal protein l7ae binding sites are present in several of these transcripts, including those encoding the anop and cbf5p components of the snorna guide complexes, these transcripts might be processed by complexes containing l7ae and possibly rnase p. seventeen of the potential snornas are antisense transcripts, of which are transcribed from dna that includes the 3′-terminus of the complementary sense gene. in cases, a snorna is transcribed convergent to an antisense rna, a gene organization also documented in s. solfataricus, n. equitans and pyrobaculum species. deep sequencing identified sense and antisense non-coding rnas in p. abyssi ge. based on a blast search, of these are also encoded in the t. kodakarensis genome, of which are clearly represented in the drna-seq libraries. a further orphan small non-coding rnas predicted and/or documented to be present in pyrococcus species and the small non-coding rnas, designated crispr rnas, predicted to be transcribed from the three crispr loci in the t. kodakarensis genome are also present in the t. kodakarensis drna-seq libraries. transcription of a crispr locus generates a long transcript that is cleaved, first releasing immature crrnas with 8 nt 5′-extensions and variable 3′-termini. these are then trimmed to produce the mature crrnas. the tss identified are fully consistent with the locations predicted for promoters within the crispr loci and with this transcript processing. orphan small non-coding rnas in addition to the orphan snornas detected and the non-coding small rnas also identified in pyrococcus species, the drna-seq libraries document the presence of previously unrecognized small transcripts, including an unrecognized snorna. orfs > codons are not present in any of these transcripts and only are conserved in other archaea arguing for t. kodakarensis specific functions. historically, computer screening for non-coding rnas in hyperthermophiles has employed a high gc content as a filter but only 30% of the non-coding rnas now identified in t. kodakarensis have gc contents >50% and only of those first reported here meet this criterion. given that the sequences of these transcripts are, on average ~43% gc, they seem unlikely to have stable base-paired secondary structures in vitro at the 85°c optimum growth temperature of t. kodakarensis but may have secondary structures stabilized in vivo by protein interactions. this is predicted for lsm binding to several srnas in h. volcanii and the t. kodakarensis genome encodes several rna-binding proteins, in addition to lsm, with alba and a putative rna-binding protein encoded by tk likely present in very high abundance.table protein-encoding genes with the highest numbers of reads per orf in the pyruvate and sulfur cdna libraries 1 exp exp 1the tkxxxx numerical gene designation for the promoter proximal gene in the transcription units that had ≥ reads. the genes are listed in decreasing order of cdna reads, with shading identifying genes present in both lists. 2the automated analysis assigned the cdna reads to genes tk tk and tk manual inspection revealed the presence of tko-sr tko-sr and tko-sr respectively, within the 5′-terminal region of each transcript. trnas, rrnas, rnase p and 7s rnas all of the trnas and rrnas annotated in the t. kodakarensis genome are present and fully covered in the drna-seq libraries. there are also antisense transcripts present complementary to six trnas and one trnathr is, itself, an antisense transcript of tk. as described above, one trnaarg appears to be cotranscribed with a snorna and a snorna encoding sequence is located immediately upstream, and is likely within the 5′-leader region and so cotranscribed with the 16s rrna-trnaala-23s rrna operon the drna-seq libraries also confirm the presence of the rnase p rna and 7s rna in t. kodakarensis cells although the 7s rna is transcribed from the dna strand opposite to that stated in the genome annotation. the drna-seq data are convincing and in agreement with the rfam database. growth and media-dependent transcription based on previous studies of p. furiosus and t. kodakarensis, we generated cdna libraries from cells growing in media with sulfur or pyruvate to increase the number of tss likely identified. we also generated cdna libraries from cells grown with sulfur to stationary phase and from cells growing with pyruvate but with sulfur added 20 min before rna isolation. the rna preparations were subjected to tex digestion before cdna synthesis and, given in vivo transcript processing and in vitro fragmentation during purification, the resulting cdna libraries were, as expected, enriched for 5′-terminal sequences. this facilitates the identification of tss but, assuming that the number of cdna reads correlates with transcript abundance, the drna-seq data also provide a semi-quantitative overview of global genome expression and are consistent with previous observations of substrate-dependent specific gene expression. based on the number of cdna reads, there is little or no transcription from ~35% of transcriptional units in cells growing in pyruvate medium, of ~28% of the tus in cells growing in sulfur medium, and of ~87% of tus in stationary phase cells. transcript abundances vary substantially, but < % are present >1000-fold above the minimal detectable level. consistent with constitutively high expression and/or transcript stability, of the most abundant transcripts were the same in rna preparations from cells growing in sulfur or pyruvate medium schut et al documented that when sulfur was added to p. furiosus cultures growing in pyruvate medium, h2s replaced h as an end-product of metabolism and transcription of the mbh operon was replaced by transcription of the mbx operon. this metabolic shift also occurs in t. kodakarensis and, based on the numbers of cdna reads obtained from rna preparations isolated from t. kodakarensis cells growing in pyruvate medium before and after sulfur addition, the transcription regulation is also conserved in t. kodakarensis. as in p. furiosus, transcription of the mbh operon that encodes the h2-generating hydrogenase is rapidly and almost fully terminated following sulfur addition and replaced by transcription of the mbx operon that encodes the h2s-generating enzyme..figure heatmap comparison of changes in gene expression after sulfur addition. changes in transcript abundance are shown, on a log2-fold scale, for the tk genes listed. comparisons were made of the cdna libraries generated from rna isolated before and 20 min after addition of sulfur to t. kodakarensis cells growing exponentially in pyruvate. values were calculated based on changes in the abundance of cdna reads, as described in materials and methods. the t. kodakarensis data are aligned and compared with the microarray hybridization results reported for sulfur-induced changes in transcription of the homologous mbx and mbh operons, and related homologous genes, in p. furiosus. the reductant needed to generate h and/or h2s is most likely supplied by a reduced ferredoxin but there are three candidate ferredoxins encoded in the t. kodakarensis genome. based on cdna reads, transcripts of tk are very abundant under all of the growth conditions investigated, indicative of ferredoxin- participating in many metabolic pathways, although there is a ~2-fold decrease after sulfur addition to pyruvate growing cells. ferredoxin- is encoded by tk the middle gene in a three gene operon that also encodes surr, a redox-responsive transcription regulator of many genes involved in sulfur metabolism. the extent of ferredoxin- reduction could provide redox-state information to surr, and so modulate surr activity, but sulfur addition had little effect on the abundance of tk1086-tk transcripts. in contrast, there was a ~6-fold decrease in tk transcripts following sulfur addition to pyruvate growing cells, arguing that ferredoxin- is likely the predominant electron donor for h production by the mbh encoded hydrogenase. intriguingly, an antisense transcript is also generated from the tk region that has increased expression in the presence of sulfur, and a surr binding site overlaps the tata box of the promoter for this antisense transcript. the antisense rna is therefore likely part of the surr regulon, and tk expression and so ferredoxin- synthesis may be indirectly subject to surr regulation through post-transcription regulation by the antisense rna. discussion and  the results obtained establish that transcription initiation occurs at > sites around the t. kodakarensis genome and recognizable bre-tata-box promoter elements are appropriately located upstream of ~78% of all the t. kodakarensis transcription units identified. as reported for other archaea, many promoters and tss are embedded within orfs, and antisense transcription occurs extensively throughout the t. kodakarensis genome adding significantly to the genome complexity, and predicting a major involvement of antisense transcripts in regulating gene expression. t. kodakarensis cells contain many small non-coding rnas, some previously identified or predicted including two candidate riboswitches but also many previously unanticipated rnas. as in p. abyssi, some have relatively au-rich sequences, in contrast to the high gc content of t. kodakarensis trnas and rrnas, and contrary to the expectation that small non-coding rnas in hyperthermophiles would be gc-rich to stabilize secondary structures. the sequences of most of these small au-rich rnas do not readily fold into canonical base-paired secondary structures, and may function as unstructured molecules, but could have secondary and tertiary structures stabilized in vivo by nucleic acid and protein interactions. most 5′-utrs in t. kodakarensis are short and some are leaderless. but, as in other archaea, there are also mrnas with long 5′-utrs that are consistent and predictive of cis-regulatory elements, although there is no direct experimental support to date for regulation in vivo by attenuators or riboswitches in archaea. the most exciting and experimentally-challenging conclusion from this, and from all other archaeal transcriptome studies to date, is that archaeal cells contain many different, often abundant and apparently non-coding small rnas. t. kodakarensis appears typical; it has a very small genome tightly packed with orfs but also with genes that encode non-translated rnas and so likely has widespread rna-based regulation. historically, rna secondary structure was sought and equated with non-coding rna function but it is now clear that such structure is not mandatory. for example eukaryotic sirnas and lncrnas apparently bind their rna and protein targets, and carry out their regulatory functions, without extensive structure. given that gene expression in archaea, and particularly transcription-related features are simpler but have many features in common with their eukaryotic counterparts, it seems likely that investigating rna-based regulation in archaea, with t. kodakarensis providing a model system, will generate results that are valuable and legitimately extrapolated into eukaryotic gene expression. 
the results identify tss for antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mrna, and confirm the presence of transcripts from all three crispr loci, the rnase p and 7s rnas, all trnas and rrnas and predicted snornas. the procedure used is designed to identify tss but, assuming that the number of cdna reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in t. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. the results identify > tss, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. sequencing cdna libraries, generated from rna isolated from cells under different growth and metabolic conditions has identified > sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. many previously unanticipated small rnas have been identified, some with relative low gc contents and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding rnas in a hyperthermophile. two putative riboswitch rnas were present in growing but not in stationary phase cells. rna sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic archaeon, thermococcus kodakarensis, under different physiological conditions.
 prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. rna sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic archaeon, thermococcus kodakarensis, under different physiological conditions.  sequencing cdna libraries, generated from rna isolated from cells under different growth and metabolic conditions has identified > sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. the primary transcription start sites upstream of annotated genes, plus primary tss and their promoters within genes, are identified. most mrnas have a 5'-untranslated region to nt long, but ~20% have 5'-utrs from to nt long and ~14% are leaderless. approximately 50% of mrnas contain a consensus ribosome binding sequence. the results identify tss for antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mrna, and confirm the presence of transcripts from all three crispr loci, the rnase p and 7s rnas, all trnas and rrnas and predicted snornas. two putative riboswitch rnas were present in growing but not in stationary phase cells. the procedure used is designed to identify tss but, assuming that the number of cdna reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in t. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. many previously unanticipated small rnas have been identified, some with relative low gc contents and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding rnas in a hyperthermophile.  the results identify > tss, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. the t. kodakarensis genome is small and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding rnas and predict extensive rna-based regulation in this model archaeon. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords transcriptomearchaeapromotersantisense rnassmall non-coding rnasriboswitchhyperthermophilehydrogen regulationissue-copyright-statement© the author 2014 