FEBS Letters 580 (2006) 6909–6914

On the interplay of gene positioning and the role of
rho-independent terminators in Escherichia coli
Nozomu Yachiea,b, Kazuharu Arakawaa,*, Masaru Tomitaa,b,cbaInstitute for Advanced Biosciences, Keio University, 5322 Endo, Fujisawa 252-8520, Kanagawa, Japan
Bioinformatics Program, Graduate School of Media and Governance, Keio University, Fujisawa 252-8520, Japanc
Department of Environmental Information, Keio University, Fujisawa 252-8520, Japan

Received 14 September 2006; revised 7 November 2006; accepted 21 November 2006
Available online 30 November 2006
Edited by Takashi Gojobori

Abstract The majority of intrinsic rho-independent terminatorsignals, reported to consist of stable hairpin structures followedby T-rich regions, possess the potential to operate bi-directionally and to induce transcription terminations on both strands ofthe DNA duplex in Escherichia coli. By using RNAMotif software, we investigated the distributions of termination motifs
around the 3 0 -ends of overlapping and non-overlapping genes at
the genomic level. We suggest that the positions of compactly
encoded E. coli genes and rho-independent terminators are optimized to terminate the adjoining genes on their antisense strands
efficiently, and not to mis-terminate overlapping transcripts, due
to their bi-directional properties.
! 2006 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.
Keywords: Rho-independent terminator; Overlapping genes;
Operon; RNAMotif; Bioinformatics

1. Introduction
In bacterial species, rho-independent terminators are widely
accepted as canonical intrinsic termination signals in the DNA
that function in minimal in vitro transcription systems [1].
Although bacterial transcription termination is controlled bymany factors, genomic analyses that have taken advantageof the availability of sequence data have suggested that mostof the transcription terminations of annotated protein-encoding and non-coding RNA transcription units are regulated
by intrinsic rho-independent terminators [1]. Intrinsic terminators are characterized by a GC-rich palindromic structure followed by a tail of A-rich region on the template DNA strand[2,3]. The palindromic region forming a stem–loop structure in
the nascent RNA is reported to pause RNA polymerases [4–6]
and weaken the interaction between elongated oligo RNA andtemplate DNA [7,8]. Owing to the weak hybridization energy
between rU and dA [9], final release may also be facilitatedby the following U-rich region [10].

*Corresponding author. Fax: +81 466 47 5099.
E-mail address: gaou@sfc.keio.ac.jp (K. Arakawa).

Abbreviations: IGR, intergenic region; tRNA, transfer RNA; rRNA,
ribosomal RNA; E. coli, Escherichia coli; nt, nucleotides; bp, base pairs

In view of these subgenomic features, many algorithms havebeen developed to identify the intrinsic terminators [11–16].
RNAMotif software is used to search RNA motifs definedby the nucleotide sequences and structural constraints given
by model file arguments named ‘descriptor’ [17]. Descriptor
files of the intrinsic terminator have been reported by Lesnik
et al. [15], based on a model consisting of stable hairpin structures followed by T-rich regions as proposed by d’Aubenton
Carafa et al. [13]. As demonstrated by Livny et al. [18], RNAMotif misses out a fraction of documented intrinsic terminator motifs detectable by other resources such as TransTerm[19]. Nevertheless, RNAMotif has been widely utilized inmany bioinformatics approaches; for example, to define
transcription terminations and to predict novel transcription
units [18,20–23]. Following these findings and applications,a bi-directional rho-independent terminator in the intergenic
region (IGR) between the tonB and P14 genes has been identified; this could function bi-directionally by sharing the twocomplementary hairpins on the double-stranded genomic
DNA, terminating the transcription of both genes [15,24].
Lesnik et al. also suggested that many bi-directional rho-independent terminators might exist in the Escherichia coli genome[15].
Here, we discuss how transcription units and intrinsic terminators are coordinately organized and distributed within
the E. coli genome based on the prediction of possible intrinsic terminator motifs at the genomic level using RNAMotif.
In order to analyze the positioning characteristics of the predicted motifs in light of the patterns of the 3 0 -ends of genes
overlapping on neighboring genes or adjoining IGRs, either
uni-directionally or bi-directionally, we constructed bioinformatics workflows using G-language Genome Analysis Environment, a generic analysis workbench for bioinformatics[25,26]. We first present that the majority of rho-independent
terminators are potentially bi-directional owing to the symmetric features of their hairpins, with low free energies inboth the genetic complements. Further, by comparing theanalysis of the 3 0 -ends of overlapping and non-overlapping
genes with those in the operon transcripts, we demonstratecomputationally the circumvention and utilization of the
intrinsic terminations associated with adjacent genes and withthe bi-directional properties of these terminators. We suggestthat the loci of intrinsic rho-independent terminators may
have been optimized to be efficiently positioned in the downstream regions of genes, compactly encoded on the E. coligenomic DNA.

0014-5793/$32.00 ! 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2006.11.053

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2. Materials and methods
2.1. Genome annotation and sequence data
The complete genome sequence of E. coli strain K-12 MG1655(RefSeq: NC000913) containing 4 639 675 bp was obtained from the
National Center for Biotechnology Information (NCBI) ftp server(ftp://ftp.ncbi.nlm.nih.gov). The annotations and positions of 4441
documented genes, including tRNA, rRNA, and other non-proteincoding RNAs, and 997 identified transcription units of E. coli were
downloaded as flat files from the EcoCyc database, Encyclopedia ofEscherichia coli K-12 Genes and Metabolism (http://www.ecocyc.org,
accessed 23 July 2006) [27].
2.2. Preparation of data on 3 0 -end positioning and operon categories
The 3 0 -ends of every gene were grouped into four categories, and thegenes included in the operon transcripts were divided into two categories. Among the 3 0 -ends of the 4441 genes, 2335 were non-overlapping
and adjoined IGRs with the adjacent downstream gene on the same
DNA strand (defined as ‘tail-to-head neighboring’), 1230 adjoined
IGRs with the adjacent downstream gene on the complementarystrand (‘tail-to-tail neighboring’), 690 overlapped with their adjacentgenes uni-directionally (‘tail-to-head overlapping’), and 186 overlapped bi-directionally (‘tail-to-tail overlapping’). Out of 977 transcription units stored in EcoCyc, 355 were prepared as operon transcripts,
each encoding more than two genes. The endmost genes of the respective operon transcripts were defined as ‘operon-end’ genes, and theremaining 884 genes were defined as ‘within-operon’ genes.
To determine the validity of our data set, which included annotatedbut experimentally unidentified genes, we repeated the same analysis
on another data set excluding genes encoding ‘hypothetical proteins’,
as annotated in GenBank, and filtered by a Gene Prediction Accuracy
Classification (GPAC) test [28] (see Supplementary Materials for details).
2.3. Prediction of rho-independent terminators
Using RNAMotif, the sequences and structural motifs of rho-independent terminators containing hairpins followed by T-rich regions
were predicted at the genomic level. The original RNAMotif model
files were constructed by machine learning of the nucleotide composition around experimentally confirmed rho-independent terminators,
which included an A-rich region at the 5 0 -side of the unidirectional
motif in addition to the experimentally suggested hairpin structurewith a 3 0 -side T-rich region, for more efficient prediction of the terminators. However, this model with the 5 0 -side A-region inherently results in preferential prediction of bi-directional terminators.
Therefore, to determine the bi-directional properties of rho-independent terminators by using unbiased criteria, we omitted the 5 0 A-region
from the model and adopted only the total DG0 scores of hairpins witha 3 0 T-region for the prediction of each motif at its optimal cut-off
score of 6!4, which is reported to significantly cover the majority ofexperimentally identified intrinsic terminators [15]. In order to compare the bi-directional properties of predicted motifs with different
scores, the predicted motifs were further prepared at three DG0 thresholds of !4, !8, and !12. The positions of transcription terminations
were defined accordingly as 7 nt upstream of the 3 0 -ends of the predicted motifs. Within the predicted terminators, those having possible
hairpin structures in their complementary DNA strands were further
screened for bi-directionality by seeking a hairpin structural motif located directly opposite of the original hairpin position; that is, symmetric bi-directional rho-independent terminators were defined if thepositions of the partial base-stacking pairs of two complementary
hairpin structures on either of the two strands coincided with eachother.
2.4. Characteristic analyses of predicted intrinsic terminators
The physical properties to potentially form stable hairpins with low
free energies reside in both DNA strands at a locus. However, pairs ofsignificant hairpin structures of the intrinsic terminators predicted by
RNAMotif in the same loci on opposite strands are not always symmetric, when considering the conditions for other sequence attributes
in the motif and/or hybridization of rU and rG. Therefore, in orderto estimate the bi-directional properties of rho-independent terminators, we performed not only the prediction of symmetric bi-directionalterminators, but also the analysis of the lengths between loop medians

N. Yachie et al. / FEBS Letters 580 (2006) 6909–6914
and termination positions of the predicted motifs and the distances
between the termination positions of two motifs encoded on the complementary DNA strands. The two-sided 95% confidence intervals of
the lengths between loop medians and termination positions of the predicted motifs were calculated. Then, for each predicted terminator, we
counted the number of antisense terminator motifs, whose termination
positions were within twice the ranges between the two-sided 95% confidence intervals upstream of the termination position in the sense
strand.
2.5. Estimation of occurrence ratio of predicted rho-independent
terminators with respect to each position around 3 0 -end of genes
Distributions of the termination positions of the predicted rho-independent terminators were analyzed bi-directionally around the 3 0 -endsof the genes within the each 3 0 -end positioning categories. For every
gene in each category, termination positions of motifs were counted
using 20-bp sliding windows with 1-bp displacement, and the motif
count at each distance (bp) was normalized and defined as ‘Ratio(%)’ by dividing by the total number of genes in the category, andby the window size, i.e. 20. In order to discuss how terminator motifs
were characteristically distributed in the each 3 0 -end positioning category, the highest ‘Ratio (%)’ of both DNA strands defined as peakratios and their relative positions from the 3 0 -end of gene were paralleled with those of the other categories.

3. Results and discussion
In this study, the optimization of gene positioning associatedwith the bi-directional properties of intrinsic rho-independent
terminators in E. coli was discussed. We initially predicted
the rho-independent terminators at genomic level by using
RNAMotif software, and suggested many of the rho-independent terminators possibly have bi-directional properties that
not only mis-terminates an overlapping transcription units
on the terminators in the same DNA strand but also thosein the complementary strand. By analyzing distributions ofpredicted rho-independent terminator motifs around the 3 0 ends of genes categorized into the four 3 0 -end positioning typesof ‘tail-to-head neighboring’, ‘tail-to-tail neighboring’, ‘tail-tohead overlapping’ and ‘tail-to-tail overlapping’, our results
suggested that the positioning of intrinsic terminators has minimized mis-termination of overlapping genes on the same
strand and the complementary strand. Furthermore, bi-directional intrinsic terminators are speculated to distribute in sucha way as to maximize co-ordinated termination of adjoining
genes on opposite strands. Though ‘within-operon’ genes are
not terminated in the proximity of downstream region of the
3 0 -ends, we indicated that the minimization of mis-termination
were independent from the biases of the ‘within-operon’ genes.
This is confirmed by observing the distribution pattern aroundthe 3 0 -ends utilizing the information of identified operon transcripts and by comparing the numbers of respective 3 0 -end
positioning type genes in the two operon categories of ‘within-operon’ and ‘operon-end’ with those of all genes.
3.1. Comparison of 3 0 -end positioning types within each operon
category
Genes in each of the 3 0 -end positioning categories werecounted and compared with respect to the corresponding operon category (Table 1). Among the total 884 ‘within-operon’genes, the numbers of ‘tail-to-head neighboring’ and ‘tailto-head overlapping’ 3 0 -ends were 611 (69.12%) and 263(29.75%), respectively. As expected, these proportions wereboth higher than ‘operon-end’ genes and all genes, because

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N. Yachie et al. / FEBS Letters 580 (2006) 6909–6914
Table 1
Numbers of genes with each 3 0 -end positioning type within the categories of ‘within-operon’, ‘operon-end’, and all genes
Positioning type of 3 0 -end

‘Within-operon’genes‘Operon-end’genes

All genes

Tail-to-head neighboring
Tail-to-tail neighboring
Tail-to-head overlapping
Tail-to-tail overlapping

611 (69.12%)
9 (1.02%)263 (29.75%)
1 (0.11%)203 (57.18%)
133 (37.46%)
8 (2.25%)
11 (3.10%)2335 (52.58%)
1230 (27.70%)690 (15.54%)
186 (4.19%)

Total

884

355

4441

the operon encodes multiple closely packed genes transcribed
together.
Only one gene of the ‘tail-to-tail overlapping’ 3 0 -end type
was found in the ‘within-operon’ category, and its proportionof 0.11% was markedly lower than the 3.10% of these genes inthe ‘operon-end’ category and the 4.19% among all genes(Table 1). The percentage of genes with ‘tail-to-tail neighboring’ 3 0 -ends (1.02%) in the ‘within-operon’ category was also
markedly lower than in the other categories (37.46% for ‘operon-end’ genes and 27.70% for all genes); 133 out of the 355‘operon-end’ genes had ‘tail-to-tail neighboring’ 3 0 -ends – thehighest proportion among those of ‘tail-to-tail neighboring’3 0 -ends in all categories. The same tendencies were observed
upon removal of the hypothetical genes (see Supplementary
Materials).
3.2. Bi-directional properties of intrinsic rho-independent
terminators
Using RNAMotif, 15 665, 5445, and 1585 rho-independent
terminators were predicted at the respective DG0 thresholds
of !4, !8, and !12. We analyzed the properties of the predicted terminators by analyzing their lengths (Fig. 1a–c), thedistances between the termination positions of two motifs encoded on the complementary DNA strands (Fig. 1d–f), and thenumbers of rho-independent terminators that were bi-directional (Fig. 1g–i).
The maximum and minimum lengths between the loop medians and termination positions of all predicted motifs shown in
Fig. 1a–c were 43 and 10.5 nt, respectively, and the average size
was 19.57 nt. The regions between the two-sided 95% confidence intervals ranged from 13 to 26 nt, from 13.5 to 26 nt,
and from 15 to 26 nt, for DG0 thresholds of !4, !8, and!12, respectively. The possible distances of the predicted termination positions in the antisense DNA strand from thosein the sense strand are displayed in Fig. 1d–f. The total frequency of antisense terminators distributed in the regions
between !52 and !26 nt away from the termination positionof terminator motif in the sense strand at DG0 threshold !4
was 133.04%. The region between positions !52 and !26 nt
was defined in accordance with the two-sided 95% confidence
interval regions from positions 13 to 26 nt as described above,and by taking twice the length to estimate the equivalent lengthof the bi-directional motifs, in order to account for the antisense structures. According to the same procedure, the totalfrequencies of antisense terminators were calculated to be
189.35% and 219.74% at DG0 thresholds !8 and !12, respectively. Within the sets of complementary terminators at DG0
scores !4, !8, and !12 and below, 3375 (21.54%), 1644
(30.19%), and 529 (33.38%), respectively, were bi-directional
rho-independent terminators, sharing double-stranded DNA
regions to form symmetrical hairpin structures with DG0 scores!4 and below. The distances between the termination posi-

Fig. 1. Characteristic analysis of predicted rho-independent terminators. Lengths between loop medians and termination positions ofpredicted rho-independent terminator motifs with DG0 scores of 6!4,!8, and !12 are displayed respectively in (a), (b), and (c). Count of
motifs at each length (nt) was normalized as ‘Ratio (%)’ by dividing by
the total number of motifs predicted at the respective threshold.
Distances of predicted transcription termination positions in theantisense DNA strand from those in the sense strand are shown in (d–f). (d), (e), and (f) represent distributions of predicted motifs in theantisense strand from motifs in the sense strands with DG0 scores of
6!4, !8, and !12, respectively. In the stacked histogram, the count ofantisense motifs at each distance (bp) was normalized as ‘Ratio (%)’ bydividing by the total number of motifs predicted at the respective
threshold. DG0 scores of antisense motifs are indicated by light gray(6!4 and >!8), gray (6!8 and >!12), and black (6!12). Counts ofbi-directional rho-independent terminators with sense strand motifs
with DG0 scores of 6!4 (g), !8 (h), and !12 (i) were also normalized
as ‘Ratio (%)’ by dividing by the total number of motifs predicted by
the respective threshold.

tions of symmetrical complementary pairs were distributed
around 40 bp. Within the putative terminators predicted by
RNAMotif, those of lower DG0 scores were suggested to havehigher bi-directional properties. This result provided the rationale that the observations of predicted terminators having low
DG0 scores were reasonable to discuss their bi-directionalities
in further analyses, conducted as follows. Although a comprehensive listing of 439 candidate bi-directional terminators has
already been reported by Lesnik et al. using a model suited forthe identification of bi-directional motifs as explained above[15], we showed here additionally using a uni-directional motif
model, that the majority of intrinsic terminator motifs with

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sufficiently low DG0 have antisense complements and thus havea high likelihood of forming bi-directional terminators. Therefore, most rho-independent terminators probably have highlybi-directional properties, as a result of the symmetry-based
low free energies and hairpin formations on the doublestranded DNA.
3.3. Optimization of E. coli gene positioning associated withintrinsic terminators
The termination positions marked by the predicted rho-independent terminators were analyzed bi-directionally around the
3 0 -ends of the genes. The distances of the terminations of theFig. 2. Distributions of rho-independent terminators around 3 0 -endsof all genes. Distances of termination positions of rho-independent
terminator motifs distributed in sense and antisense DNA strands fromthe 3 0 -ends of genes are shown in (a) and (b), respectively. Frequencyof the motifs with respect to the position around 3 0 -end of genes werenormalized as ‘Ratio (%)’ (for details, see the Section 2.5). DG0 scores
are indicated by light gray (6!4 and >!8), gray (6!8 and >!12), andblack (6!12) bars in the stacked histogram. From the histograms, bidirectional rho-independent terminators (not colored by motif scores)
were extracted to (c) sense strand and (d) antisense strand.

N. Yachie et al. / FEBS Letters 580 (2006) 6909–6914

predicted terminators and the bi-directional terminators fromthe 3 0 -ends of all genes in both DNA strands are shown in
Fig. 2a–d, including certain fractions of those having lower
DG0 scores of 6!8 and 6!12, and their bi-directional properties were also suggested by focusing on the downstream partsof the 3 0 -ends of the genes. The predicted termination positions
were distributed at around +33 bp with the peak ratio of 3.92%
in the sense strand, and at about !4 bp with the peak ratio of
2.66% in the antisense strand, as calculated from the normalized count per gene per position (for details, see Section 2.5).
The terminations of bi-directional terminators were distributedat around +34 bp in the sense strand, where the peak ratio was
1.53%; in the antisense strand they were distributed at around
0 bp, where the peak ratio was 1.23%.
Similarly to the result for all genes, the termination positions
were distributed around the ‘tail-to-head neighboring’ and‘tail-to-tail neighboring’ 3 0 -ends (Fig. 3a–d and e–h). The peakratios of 3.66% (1.09% bi-directional) in the sense strand and1.36% (0.79% bi-directional) in the antisense strand, calculatedusing all predicted terminators, were distanced +34 bp (+37 bp
bi-directional) and !4 bp (+2 bp bi-directional), respectively,from the 3 0 -ends of the genes in the ‘tail-to-head neighboring’category. Likewise, for the ‘tail-to-tail neighboring’ category,the peak ratios were 7.07% (3.63% bi-directional) in the sense
strand and 6.86% (3.19% bi-directional) in the antisense
strand, distanced +32 bp (+32 bp bi-directional) and !4 bp(0 bp bi-directional) from the 3 0 -ends of the genes. The peakratios of the ‘tail-to-tail neighboring’ category, including higher fractions of lower DG0 scores of 6!8 and 6!12, were significantly higher than those of the ‘tail-to-head neighboring’and of all genes (Fig. 2), suggesting the marked adoption ofbi-directional properties that can terminate a pair of ‘tail-totail neighboring’ genes with one shared bi-directional terminator.

Fig. 3. Distributions of rho-independent terminators around 3 0 -ends of ‘tail-to-head neighboring’ (a–d), ‘tail-to-tail neighboring’ (e–h), ‘tail-to-headoverlapping’ (i–l), and ‘tail-to-tail overlapping’ (m–p) genes. For details, see caption to Fig. 2.

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N. Yachie et al. / FEBS Letters 580 (2006) 6909–6914

In the categories of ‘tail-to-head overlapping’ and ‘tail-totail overlapping’, marked distributions of terminators were
not observed, compared with those described above (Fig. 3i–l
and m–p). We suggest that the result in the ‘tail-to-head overlapping’ category, showing a circumvention of the terminatorstructures, might be designed to prevent mis-termination,
pausing the transcription in the middle of another overlapped
gene in co-orientation. Genes in this category may be terminated by other intrinsic motifs that are not given in the model
used here, or by other transcription termination factors [1].
However, a comparison of the ‘within-operon’ and ‘operonend’ categories (Fig. 4) showed that the ‘within-operon’ category also lacked marked distributions, and many of the ‘tailto-head overlapping’ genes were suggested to be involved in
the operon transcripts (see Table 1 for details). We thus speculate that some of the ‘tail-to-head overlapping’ genes are‘within-operon’ genes, and therefore are not terminated at
the proximal downstream regions of their 3 0 -ends.
From the results of the ‘tail-to-tail overlapping’ category, we
further consider that the uni-directional intrinsic motifs terminating the ‘tail-to-tail overlapping’ genes are avoided to decrease the chance of mis-termination of their adjacent genes
in antisense strands by their possible bi-directional properties.
Only a negligible number of genes in this category belonged tothe ‘within-operon’ category (Table 1), and therefore somekind of termination other than by the intrinsic signal is expected – for example, by rho-dependent termination [1]. Negative selective pressure works against gene formation in theantisense strands of operon transcripts (Table 1), and thismay be partly attributable to the circumvention of mis-termination of the antisense gene caused by the bi-directional terminator. On the other hand, in the analyses of the ‘operon-end’category, the high peak ratios of 8.07% (3.66% bi-directional)of the sense strand and 4.19% (2.75% bi-directional) of theantisense strand were distanced at +32 bp (+34 bp bi-direc-

tional) and !2 bp (+5 bp bi-directional) from the 3 0 -ends ofthe genes (Fig. 4); we considered that this result was causedby the inclusion of many ‘tail-to-tail neighboring’ genes inthe ‘operon-end’ category, as suggested in Table 1, or was
due to the high sensitivity of the experimentally confirmed
transcription terminations.
The same procedures were repeated for every positioning
pattern of the 3 0 -ends after filtering by the GPAC test andfor randomly sampled sets of 100 genes by a bootstrap test,
but the results showed no marked change (see Supplementary
Materials). We therefore conclude that of our results were notbiased by variations in the annotation accuracy of genes or by
the numbers of genes in the respective categories.
We conducted a bioinformatics analysis to investigate gene
positioning in relation to the intrinsic termination motifs,
and we discussed here the optimal positioning of bi-directionalterminators in utilizing efficient terminations of head-on andnon-overlapping gene pairs in a dual role and in circumventing
the mis-termination of overlapping transcripts. A previous
study has indicated that such structure-dependent intrinsic terminators appear to be employed in only a few bacterial species,
as suggested by the fact that the calculation of free energies
around the 3 0 -ends of their genes resulted in the lowest distributions in regions downstream of ‘tail-to-tail neighboring’genes [29]. We also suggest that our data support the conceptof dynamic gene positioning by a combination of intrinsic terminators that has possibly been optimized in E. coli.
Acknowledgements: The authors are grateful to the members of MGSP
at the Institute for Advanced Biosciences, Keio University, for theircritical discussions. This research was supported in part by the Japan
Society for the Promotion of Science (JSPS).

Appendix A. Supplementary data
Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.febslet.2006.11.053.
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Fig. 4. Distributions of rho-independent terminators around 3 0 -endsof ‘operon-end’ (a–d) and ‘within-operon’ (e–h) genes. For details, seecaption to Fig. 2.

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