Minirevie w

          THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 270. No. 42, Issue of Ortober 20, pp 24623-24626. 1995
                     Printed in USA.

Unusual Reverse Transcriptases*

Maxine F. Singer$

From the Carnegie Institution of Washington,
Washington, D. C. 20005 and the Laboratory of
Biochemistry, NCI, National Institutes of Health.
Bethesda, Maryland 20892

Reverse transcriptase (RTase) ,' rather than being unique to
retroviruses as once appeared likely, is encoded in many DNA
genomes (1, 2). RTase genes occur within 1) the retrovirus-like
class I retrotransposons that have long terminal repeats (e.g
Ty of yeast), 2) class I1 (or LINE-like) retrotransposons that
lack terminal repeats of any kind and typically have a dA-rich
stretch at the 3`-end of the sense DNA strand, and 3) unusual
retroelements of chromosomal and organellar origin. Telomer-
ase is also an RTase (3). Molecular phylogenetic analyses indi-
cate that the RTases encoded by the class I1 retrotransposons
and retroelements (called here type 2 RTases) are more like one
another in predicted amino acid sequence than they are
like the RTases (type 1) encoded by retroviruses and class I
retrotransposons (4 -7).
Soon after the discovery of the coding sequences predicting
the type 2 RTases, many investigators recognized that these
enzymes would be mechanistically distinctive from the already
well known type 1 enzymes (8 -1 2); the complex series of events
that assure replication of long terminal repeats, including in-
ternal primer binding sites on the RNA template, priming by
tRNAs. and template switching, is unnecessary. Another dif-
ference between type 1 and type 2 RTases is the apparent
absence, in most type 2 coding sequences, of segments that
predict an RNase H. As summarized in this review, a few type
2 enzymes and telomerase have now been studied and, remark-
ably, each of them has a distinctive priming mechanism. More-
over, none of them appears to utilize as primer a nucleotide
covalently bound to the RTase protein as does the type 1
hepatitis B virus RTase (13).

The RTases of LINE-like Retrotransposons

The RZBm RTase-Two types of LINE-like elements, R1 and
R2. are found inserted at specific positions in some copies of the
28 S rRNA genes of many insect species (14, 15). An Esche-
richia coli expression plasmid containing the entire single open
reading frame (OW) (predicting a 123-kDa protein) of the
Bombyx mori R2 element (R2Bm) produces a 120-kDa protein
with integrase (endonuclease) and RTase activity. The inte-
grase produces staggered, site-specific double strand breaks at
the observed insertion site in 28 S rDNA yielding 5'-phosphate
and 3'-hydroxyl termini and 2-base pair long 5'-overhangs (16,
17): the initial nick is between a C and an A residue (5'-(A/C)C-)
on the 28 S rDNA coding strand. RNA is required for optimal
activity. In the presence of RNA, the integrase rapidly cata-
lyzes the initial nick to form relaxed plasmid circles and then

* This minireview will be reprinted in the 1995 Minireview Compen-
dium, which will be available in December, 1995.
t To whom correspondence should be addressed: 1530 P Street, N. W..
Washington, D. C. 20005. Tel.: 202-387-6404: Fax: 202-462-7395:
E-mail: msinger@science.pst.ciw.edu.

The abbreviations used are: RTase. reverse transcriptase: ORF,
open reading frame: nt. nucleotide(s); RNP, ribonucleoprotein; msDNA.
multicopy single-stranded DNA; msdRNA. RNA attached to msDNA.

slowly converts these to full-length linear duplexes. However,
in the absence of RNA only the nicked, relaxed circles are
produced.
When dNTPs and in vitro synthesized R2Bm RNA are added
to the integrase reaction mixtures, reverse transcription occurs
and is dependent upon the presence of the rDNA target site.
Analysis of the cDNA product demonstrated that the 3`-hy-
droxyl at the first nick in the DNA target is the primer for the
RTase; the template is R2Bm RNA (17) (Fig. la). These find-
ings indicated that target site cleavage and reverse transcrip-
tion are coupled, as predicted by early models for LINE-like
element insertion (9 -1 2).
The relative rates at which the reaction products accumulate
are as expected if second strand cleavage follows reverse tran-
scription. Thus, the initial product is a branched chain in which
the cDNA copy of the R2Bm RNA is covalently linked to the 28
S rDNA and hydrogen bonded to the RNA. Significantly, al-
though an RNA chain lacking the R2Bm sequences can act as
cofactor for second strand cleavage, only RNAs containing the
3'-end of R2Bm RNA can serve as the template for the RTase;
it appears that sequences within the 250-nt long 3'-untrans-
lated region of R2Bm RNA are recognized by the enzyme and,
together with 5-10 residues in the primer end of the 28 S
rDNA, permit chain synthesis (18).
All known genomic R2Bm elements have 4 As at the 3'-
junction with 28 S rDNA. Experiments with in vitru synthe-
sized R2Bm RNA containing varying numbers of terminal As
indicate that the efficiency of cDNA synthesis decreases as the
terminus changes from 4 to 1 to 8 As (17. 18). Substitution of
the four As with other bases can yield efficient templates; many
of the cDNA products initiate at the RNA terminal residue.
When the (A)4 terminus is reduced in length, occasional inter-
nal initiations and frequent additions of extra nontemplated
residues occur. These observations may reflect constraints im-
posed by the necessity of the enzyme to position itself accu-
rately on both the RNA template and the 28 S rDNA (18).
Virtually nothing is known about second strand DNA synthesis
or the fate of the RNA template except that the two overhang-
ing bases left after cleavage of the second 28 S rDNA strand are
removed.
RTases of Other Class II Retrutransposons-Several other
LINE-like elements have been shown to encode active RTases,
but the reaction mechanisms have not been studied. The coding
region of the Drosophila melanogasterjockey element yields, in
E. coli, active RTase associated with a 92-kDa polypeptide (19).
RTase activity, accompanied by L 1Hs RNA, has been reported
to occur in the microsomal fraction of human NTeraPDl cells,
but it remains to be proven that it is encoded by LlHs (20). In
more definitive experiments, LlHs (21) or CREl (of Crithidia
fasciculata) (22) RTase was detected in partly purified virus-
like particles formed in yeast cells transformed with class I
retrotransposon Tyl in which the RTase gene was replaced by
the LlHs or CREl RTase coding region. The RTases encoded by
LlHs (23). CRE1,` and R2Bm3 have also been detected with an
indirect genetic assay for the RTase-dependent formation, in
yeast, of processed pseudogenes (24). In the case of LlHs, the
complex structures of the newly inserted sequences suggest

' A. Gabriel, personal communication.

T. Eickbush. personal communication.

24623

24624

Minireview: Unusual Reverse Transcriptases

RZ&lfl Maurs;lcuillt. Ptyrinid Prok%vic Rclt&i Tclr)rmrBse

id@ nom &%'t~thW%l

FIG. 1. Schematic representations
of four RTase reactions described in
the text: a, R2Bm; b, Mauriceville
plasmid; c, bacterial retron; d, telom-
erase. Red is RNA, black is DNA, and
green is newly synthesized DNA. Refer to
the text for explanations and references.

H

that the RTase readily switches from one template to another
(23).
     The Prokaryotic Group of RTases

A subset of type 2 RTases can be defined based on the
similarity of predicted amino acid sequences (6, 7). This subset
includes the enzymes encoded by mitochondrial plasmids found
in some strains of Neurospora, bacterial retrons, and group I1
introns and is called the prokaryotic group, reflecting the pro-
karyote origin of mitochondria and chloroplasts (7, 25).
The de Novo Synthesis of DNA by the RTases of a Neurospora
Mitochondrial Plasmid-The Mauriceville plasmid (3.6 kilo-
base pairs) found in Neurospora mitochondria encodes a 710-
amino acid long polypeptide in its single ORF (25). Ribonucle-
oprotein (RNP) particles isolated from the mitochondria
contain full-length, plus strand transcripts of the plasmid and
RTase activity associated with a homodimer of the 81-kDa
protein that is encoded by the ORF (25, 26). The RTase within
the RNPs copies the endogenous RNA to yield an RNA-cDNA
duplex with a full-length (minus strand) cDNA (26, 27). After a
lag, plus strand DNA is synthesized: an RNase H present in the
RNP but not encoded in the plasmid can degrade the RNA (28).
The 3'-end of the plasmid transcript has striking similari-
ties, in sequence and secondary structure, to the tRNA-like 3'
termini of plant RNA virus genomes including the CCA 3'
termini (Fig. lb) (25, 29). And like the RNA-dependent RNA
polymerases encoded by RNA viruses, many of the (minus
strand) cDNAs synthesized in the RNPs begin (5'-end) with a G
corresponding to the penultimate C in the RNA (27). Another
large group of the cDNAs utilize DNA primers unrelated to the
3'-end of the RNA template and begin copying from the 3'-
terminal A in the template. Experiments with purified RTase
clarified these findings.
The RTase can be released from the RNPs with micrococcal
nuclease and purified (30). When partially purified enzyme was
incubated with an RNA template synthesized in vitro and
containing 5'-truncated plasmid sequences, the cDNA products
were about 20 nucleotides longer than the template RNA. The
extra nucleotides were at the 5'-end of the cDNA and derived
from priming oligodeoxynucleotides that were bound to the
RTase. The primers were each different in sequence and
length, and most appeared to be short cDNAs copied from
plasmid or mitochondrial RNA. Moreover, these primers were
all joined directly to the 5'-TGG sequence copied from the
3'-ACC end of the template RNA (as were some of the primed
cDNAs synthesized within RNPs and described above) and

could be cleaved from the RNA-DNA duplex with S1 nuclease.
Assuming that the plasmid RTase catalyzed synthesis of these
primers, then it seems that the enzyme is capable of switching
templates, in analogy with the type 1 RTases.
When the RTase is freed of the bound primers by treatment
with polyethyleneimine (31), it can utilize either exogenously
supplied oligodeoxynucleotides or the 3'-end of the RNA tem-
plate itself as primer; that is, the primer can be either DNA or
RNA. Most interestingly, however, the RTase is also efficient in
de novo cDNA synthesis (Fig. 1 b) and is the first DNA polym-
erase known to initiate DNA chains. A template containing
only the 3'-terminal 26 residues of the plasmid RNA is suffi-
cient to direct specific, de novo initiation, primarily opposite the
penultimate C residue (as in the other major group of cDNAs
synthesized in RNPs). The 5'-terminal G residue can be sup-
plied by free deoxyguanosine, dGMP. or dGTP. If the 3'-termi-
nal residues of the template are missing or if extra nucleotides
are added to the 3'-end, copying occurs but not de novo synthe-
sis: instead, the 3'-end of the RNA serves as a primer. The
similarity between this RTase and the RNA-dependent RNA
polymerases of QP and brome mosaic virus is underscored by
their common ability to recognize the 3'-terminal tRNA-like
structure of the template and initiate synthesis by copying the
penultimate residue.
RTases of Bacterial Retrons-Retrons. so-called by Temin
(32). have been described in the chromosomes of myxobacteria,
a few strains of E. coli, and several other bacteria (33-35).
These DNA elements (from 1.3 to 3 kilobase pairs long) vary in
detailed structure and sequence, but all include a single chro-
mosomal transcription unit containing, from 5' to 3', sequences
encoding msdRNA. msDNA, and RTase. Current understand-
ing of the RTase is derived from structural analysis of the in
vivo products and in vitro analysis of a retron RTase purified to
homogeneity after expression from a recombinant plasmid in E.
coli (36). It was the discovery and characterization of the prod-
uct of the reverse transcription, msDNA (for multicopy single-
stranded DNA), that led to the discovery of the RTase.
The msDNAs in various bacteria differ in length and se-
quence but share common features (Fig. IC, bottom): 1) the
5'-end of the DNA (65-163 nt) is linked, through a 2',5'-phos-
phodiester, to a guanosine within a short RNA (fewer than 100
nt), the msdRNA; 2) from 6 to 8 nt at the 3'-end of both
the msDNA and msdRNA are complementary and form a
duplex; and 3) both msDNA and msdRNA assume stable
secondary structures.

Minireview: Unusual Reverse Transcriptases 24625

Although the retron RTases vary in size from about 300 to
590 amino acids (34. 35). the enzyme in cell extracts sediments
in association with msDNA as a large complex and has an
apparent molecular mass of 600 -700 kDa on molecular sieves
(33). The folded RNA of the retron transcription unit provides
both the template and primer for the enzyme (Fig. 1 c, top) and
may also serve as mRNA for RTase translation although this
has not been proved. Among the important features of the
folded RNA is a duplex stem formed by the inverted repeats a2
and a1 that bracket the msdRNA and msDNA region of the
retron transcript and several stendloop structures. A G residue
in the short single-stranded msdRNA segment just 3' of the
a2.al stem provides the Z'-hydroxyl that primes reverse tran-
scription. The residue in the msDNA region that marks the
start of the template segment also occurs just after the end of
the a2al duplex. Thus, the al-a2 stem brings the primer and
the template in close proximity. With the purified enzyme and
the folded RNA as template, msDNA is synthesized and its
5`-end is linked to the Z'-hydroxyl of the expected guanosine
residue in the msdRNA region (36). Elongation of the DNA
proceeds along the template, and the efficiency of chain exten-
sion depends on the absence of any stable secondary structures,
but not on the particular sequence, within the msDNA tem-
plate region (37). In most instances, copying stops and the
chain terminates after addition of the 6-8 nucleotides that
form the duplex between the 3'-ends of the msdRNA and
msDNA segments; the mechanism of this specific chain termi-
nation has not been determined. In contrast to the lack of
specificity for the RNA sequence in the msDNA template re-
gion, the msdRNA region must be from the same retron as the
RTase; when the msdRNA regions of two different retrons are
exchanged, msDNA synthesis does not occur (38).
With one possible exception, the RTase coding regions of
retrnns do not predict RNase H segments. Nevertheless, the
formation of msDNA by the proposed scheme requires the
removal of the RNA in the msDNA region of the template.
Experiments with E. coli cells carrying mutations in chromo-
somal RNase H genes indicate that cellular RNase H is likely to
play a role in msDNA synthesis (39).
The RTase ofa Group II Intron-Group I1 introns were first
found in the genomes of fungal and plant organelles (40) and
more recently in certain cyanobacteria and proteobacteria (4 1).
They have attracted attention because of 1) their ability to
self-splice in vitro (though apparently not in vivo), 2) a splicing
mechanism like that used by eukaryotic nuclear genes, 3) their
mobility, 4) the presence, in some of them, of coding regions for
genetically defined proteins, maturases. that are required for
in vivo splicing, and 5) the presence, in some of them (group
IIA). of a coding region that predicts a RTase. RNPs isolated
from yeast mitochondria support the synthesis of cDNAs com-
plementary to RNA transcribed from the coxl mitochondrial
gene and containing sequences from the first and second (group
IIA) introns in the gene, a11 and a12 (42). The a12 enzyme can
also utilize poly(rA)-oligo(dT). Analysis of mutant alleles of
coxl indicated that RTase activity depends on the ORF of
either a11 or aI2, depending on the strain. The cDNAs initiated
by a12 RTase start at multiple sites near the 3'-end of a12 or
within exon 3, and their structures are such that the templates
were either excised introns or unspliced or partially spliced
mRNAs. Neither the priming mechanism nor the template
requirements are understood, although the cDNA structures
would be consistent with de novo chain initiation.

Telomeres and Telomerase

The formation of telomeric structures is associated with
RTase in two known ways. Telomerase. which adds character-
istic G-rich repeats to the ends of chromosomes in many eu-

karyotes, is an RTase (3), and D. melanogaster`s distinctive
telomeric structures are made up of multiple copies of two
LINE-like elements, HeT-A (43, 44) and TART (45, 46), both of
which can transpose onto Drosophila telomeres.
Telomerases are RNPs containing an uncapped RNA that
acts as template (Fig. 1 d). In the purified Tetrahymena ther-
mophila enzyme, 80- and 95-kDa polypeptides bind the RNA
and the telomeric primer DNA, respectively (47). There is lim-
ited homology between the amino acid sequences predicted
from the cloned gene (and cDNA) for p95 and viral RNA-de-
pendent RNA polymerases: otherwise, the telomerase proteins
are different from other RTases and from each other (47). The
telomerase RNAs vary in length (from 159 nt in Tetrahymena
to 1300 in yeast (48)) and sequence, but each includes a se-
quence complementary to the species-specific, 3` single strand
overhanging, G-rich telomeric repeat; a conserved secondary
structure among the protozoan telomerase RNAs includes a
generally single-stranded, although partly constrained, config-
uration for the template region (49 -5 1). Active enzyme can be
reconstituted after removal of the RNA by nuclease treatment
(47, 52).
In the first step in telomere extension, the 3' G-rich overhang
base pairs with a few nucleotides in the telomerase RNA (Fig.
1 d). The RTase then copies the rest of the basic repeat unit (e.g.
GGGTTG in Tetrahymena) and pauses and shifts relative to
the telomere so that the new terminus can be repositioned for
addition of the next repeat (3, 52). The mechanism whereby
copying pauses and the primer shifts once the repeat segment
is complete is unknown: as already mentioned, a similar ques-
tion arises in the synthesis of retron msDNA. Other experi-
ments indicate that the rate and processivity of polymerization
in vitro are strongly dependent on the nucleotides just 5' of the
terminal G-rich repeat on the primer (53, 54): it is likely that
this represents a secondary binding site, the anchor site, for
p95 and that such binding contributes to processivity.

Comments

Recent discussions about the origin of life on Earth postulate
an early, "RNA world and the conversion of that RNA world to
one in which genetic information is stored in DNA through the
mediation of a primitive RTase (55-57). Thus, special interest
has focused on the widespread type 2 RTases. It has been
suggested that they may 1) have evolved from a primitive
RNA-dependent RNA polymerase such as those now associated
with some RNA viruses, 2) represent the most primitive
RTases yet observed and thus may be ancestral to type  1
RTases and DNA polymerases, and 3) be the closest relatives
we know to the RTase required by the "RNA world" hypothesis

Molecular phylogenies suggest that all the type 2 RTases
may be more closely related than the organisms that harbor
them and thus have, in part, an independent evolutionary
history (4 -7). This could be the consequence of conservation of
the RTase coding sequence within the separate genomes over
long periods of evolutionary history or could reflect horizontal
transfer of elements across species and even phyla. There is
evidence for horizontal transfer of jockey among distantly re-
lated members of the Drosophila genus (58). Apart from the
similar RTase coding sequences, certain other features recur
among the type 2 RTases and even telomerase. These include
the importance of template secondary structure, the recogni-
tion by the enzymes of the 3'-terminal structure of the RNA
template, the initiating role of guanosine nucleotides, and the
association of the enzymes with RNPs. Thus, while each of the
enzymes has evolved to develop distinctive properties, their
similarities hint at a common ancestor.

(27, 31, 55-57).

24626

Minireview: Unusual

Acknowledgments-I thank M. Inouye, A. Lambowitz. and H.
Kazazian for providing preprints of manuscripts and T. Eickbush
and A. Gabriel for unpublished information. I am grateful to
Edward Kuff, Claude Klee, Andrew Clements. and Fang-Min Sheen for
reviewing the manuscript.

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