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From the Reverse transcriptase (RTase), ( 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, 9, 10, 11, 12) ; the
complex series of events that assure replication of long terminal
repeats, including internal primer binding sites on the RNA template,
priming by tRNAs, and template switching, is unnecessary. Another
difference 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, remarkably, each of them has a distinctive priming
mechanism. Moreover, 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
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`-hydroxyl at the first nick in the DNA target is the primer for the
RTase; the template is R2Bm RNA (17) (Fig. 1a). These findings indicated that
target site cleavage and reverse transcription are coupled, as
predicted by early models for LINE-like element
insertion(9, 10, 11, 12) .
Figure 1:
Schematic
representations of four RTase reactions described in the text: a, R2Bm; b, Mauriceville plasmid; c,
bacterial retron; d, telomerase. Red is RNA, black is DNA, and green is newly synthesized DNA.
Refer to the text for explanations and
references.
The
relative rates at which the reaction products accumulate are as
expected if second strand cleavage follows reverse transcription. 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, although 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`-untranslated
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 vitro synthesized 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)
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 II introns and is called the prokaryotic group, reflecting the
prokaryote origin of mitochondria and
chloroplasts(7, 25) .
The 3`-end of the plasmid
transcript has striking similarities, in sequence and secondary
structure, to the tRNA-like 3` termini of plant RNA virus genomes
including the CCA 3` termini (Fig. 1b) (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 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 template 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. 1b) and is the first DNA
polymerase known to initiate DNA chains. A template containing only the
3`-terminal 26 residues of the plasmid RNA is sufficient 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 supplied by free
deoxyguanosine, dGMP, or dGTP. If the 3`-terminal residues of the
template are missing or if extra nucleotides are added to the 3`-end,
copying occurs but not de novo synthesis; instead, the 3`-end
of the RNA serves as a primer. The similarity between this RTase and
the RNA-dependent RNA polymerases of Q
The msDNAs in various bacteria differ in
length and sequence but share common features (Fig. 1c, bottom): 1) the 5`-end of the DNA (65-163 nt) is linked,
through a 2`,5`-phosphodiester, 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. 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. 1c, 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 stem/loop structures. A G residue in the short single-stranded
msdRNA segment just 3` of the a2 With one possible exception, the RTase coding regions of retrons 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 chromosomal RNase H genes indicate that cellular RNase H
is likely to play a role in msDNA synthesis(39) .
The formation of telomeric structures is associated with
RTase in two known ways. Telomerase, which adds characteristic G-rich
repeats to the ends of chromosomes in many eukaryotes, 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. 1d). In the
purified Tetrahymena thermophila enzyme, 80- and 95-kDa
polypeptides bind the RNA and the telomeric primer DNA,
respectively(47) . There is limited homology between the amino
acid sequences predicted from the cloned gene (and cDNA) for p95 and
viral RNA-dependent 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 sequence
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, configuration for the template
region(49, 50, 51) . 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. 1d). 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 question arises in the synthesis of
retron msDNA. Other experiments 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. 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, 56, 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(27, 31, 55, 56, 57) . 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, 5, 6, 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 related 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 recognition 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.
Volume 270,
Number 42,
Issue of October 20, 1995 pp. 24623-24626
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
INTRODUCTION
The RTases of LINE-like Retrotransposons
The Prokaryotic Group of RTases
Telomeres and Telomerase
Comments
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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 II (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. Telomerase
is also an RTase(3) . Molecular phylogenetic analyses indicate
that the RTases encoded by the class II 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, 5, 6, 7) .
The R2Bm 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 Escherichia coli expression plasmid containing the entire
single open reading frame (ORF) (predicting a 123-kDa protein) of the Bombyx mori R2 element (R2Bm) produces a 120-kDa protein with
integrase (endonuclease) and RTase activity. The integrase 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 catalyzes the initial nick to form relaxed
plasmid circles and then slowly converts these to full-length linear
duplexes. However, in the absence of RNA only the nicked, relaxed
circles are produced.
terminus is reduced in length,
occasional internal initiations and frequent additions of extra
nontemplated residues occur. These observations may reflect constraints
imposed by the necessity of the enzyme to position itself accurately 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 overhanging bases left after cleavage of
the second 28 S rDNA strand are removed.RTases of Other Class II Retrotransposons
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 melanogaster jockey element yields, in E. coli, active RTase associated with a 92-kDa polypeptide(19) .
RTase activity, accompanied by L1Hs RNA, has been reported to occur in
the microsomal fraction of human NTera2D1 cells, but it remains to be
proven that it is encoded by L1Hs(20) . In more definitive
experiments, L1Hs (21) or CRE1 (of Crithidia
fasciculata) (22) RTase was detected in partly purified
virus-like particles formed in yeast cells transformed with class I
retrotransposon Ty1 in which the RTase gene was replaced by the L1Hs or
CRE1 RTase coding region. The RTases encoded by L1Hs(23) ,
CRE1, (
)and R2Bm (
)have also been detected with
an indirect genetic assay for the RTase-dependent formation, in yeast,
of processed pseudogenes(24) . In the case of L1Hs, the complex
structures of the newly inserted sequences suggest that the RTase
readily switches from one template to another(23) .
The de Novo Synthesis of DNA by the RTases of a Neurospora
Mitochondrial Plasmid
The Mauriceville plasmid (3.6 kilobase
pairs) found in Neurospora mitochondria encodes a 710-amino
acid long polypeptide in its single ORF(25) . Ribonucleoprotein
(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) .
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.
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, 34, 35) . These DNA elements
(from 1.3 to 3 kilobase pairs long) vary in detailed structure and
sequence, but all include a single chromosomal transcription unit
containing, from 5` to 3`, sequences encoding msdRNA, msDNA, and RTase.
Current understanding 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 product of the reverse
transcription, msDNA (for multicopy single-stranded DNA), that led to
the discovery of the RTase.a1 stem provides the 2`-hydroxyl
that primes reverse transcription. The residue in the msDNA region that
marks the start of the template segment also occurs just after the end
of the a2a1 duplex. Thus, the a1a2 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 2`-hydroxyl of the expected guanosine residue in the msdRNA
region(36) . Elongation of the DNA proceeds along the template,
and the efficiency of chain extension depends on the absence of any
stable secondary structures, but not on the particular sequence, within
the msDNA template 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 termination has
not been determined. In contrast to the lack of specificity for the RNA
sequence in the msDNA template region, 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) . The RTase of a Group II Intron
Group II introns
were first found in the genomes of fungal and plant organelles (40) and more recently in certain cyanobacteria and
proteobacteria(41) . 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
complementary to RNA transcribed from the cox1 mitochondrial
gene and containing sequences from the first and second (group IIA)
introns in the gene, aI1 and aI2(42) . The aI2 enzyme can also
utilize poly(rA)-oligo(dT). Analysis of mutant alleles of cox1
indicated that RTase activity depends on the ORF of either aI1 or aI2,
depending on the strain. The cDNAs initiated by aI2 RTase start at
multiple sites near the 3`-end of aI2 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.
)))
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 Fong-Min Sheen for reviewing the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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