scholarly journals Interactions between Ty1 Retrotransposon RNA and the T and D Regions of the tRNAiMet Primer Are Required for Initiation of Reverse Transcription In Vivo

1998 ◽  
Vol 18 (2) ◽  
pp. 799-806 ◽  
Author(s):  
S. Friant ◽  
T. Heyman ◽  
A. S. Byström ◽  
M. Wilhelm ◽  
F. X. Wilhelm
Keyword(s):  
Reverse Transcription ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Genomic Rna ◽  
Complementary Sequences ◽  
Compensatory Mutations ◽  
Ty1 Retrotransposon ◽  
Ty1 Retrotransposition ◽  
Strong Stop

ABSTRACT Reverse transcription of the Saccharomyces cerevisiaeTy1 retrotransposon is primed by tRNAi Met base paired to the primer binding site (PBS) near the 5′ end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNAi Met. A structural probing study of the interactions between the Ty1 RNA template and the tRNAi Met primer showed that besides interactions between the PBS and the 3′ end of tRNAi Met, three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNAi Met. To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNAi Metwere tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNAi Met contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNAi Met and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNAi Met.

scholarly journals Replication Errors during In Vivo Ty1 Transposition Are Linked to Heterogeneous RNase H Cleavage Sites

1998 ◽  
Vol 18 (2) ◽  
pp. 1094-1104 ◽  
Author(s):  
Emilie H. Mules ◽  
Ozcan Uzun ◽  
Abram Gabriel
Keyword(s):  
Rnase H ◽  
Primer Binding Site ◽  
Cleavage Sites ◽  
Minus Strand ◽  
Terminal Sequence ◽  
Cdna Synthesis ◽  
Replication Errors ◽  
Key Points ◽  
Strong Stop

ABSTRACT We previously identified a mutational hotspot upstream of the Ty1 U5-primer binding site (PBS) border and proposed a novel mechanism to account for this phenomenon during Ty1 replication. In this report, we verify key points of our model and show that in vivo RNase H cleavage of Ty1 RNA during minus-strand strong-stop synthesis creates heterogeneous 5′ RNA ends. The preferred cleavage sites closest to the PBS are 6 and 3 bases upstream of the U5-PBS border. Minus-strand cDNA synthesis terminates at multiple sites determined by RNase H cleavage, and DNA intermediates frequently contain 3′-terminal sequence changes at or near their template ends. These data indicate that nontemplated terminal base addition during reverse transcription is a real in vivo phenomenon and suggest that this mechanism is a major source of sequence variability among retrotransposed genetic elements.

scholarly journals Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

2002 ◽  
Vol 278 (10) ◽  
pp. 8006-8017 ◽  
Author(s):  
Yan Chen ◽  
Mini Balakrishnan ◽  
Bernard P. Roques ◽  
Philip J. Fay ◽  
Robert A. Bambara
Keyword(s):  
Reverse Transcription ◽  
Minus Strand ◽  
Hiv 1 ◽  
Strong Stop

scholarly journals Pausing during Reverse Transcription Increases the Rate of Retroviral Recombination

2006 ◽  
Vol 80 (5) ◽  
pp. 2483-2494 ◽  
Author(s):  
Christian Lanciault ◽  
James J. Champoux
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Reverse Transcription ◽  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Primer Binding Site ◽  
Infection Assay ◽  
Green Fluorescent

ABSTRACT Retroviruses package two copies of genomic RNA into viral particles. During the minus-sense DNA synthesis step of reverse transcription, the nascent DNA can transfer multiple times between the two copies of the genome, resulting in recombination. The mechanism for this process is similar to the process of obligate strand transfers mediated by the repeat and primer binding site sequences. The location at which the DNA 3′ terminus completely transfers to the second RNA strand defines the point of crossover. Previous work in vitro demonstrated that reverse transcriptase pausing has a significant impact on the location of the crossover, with a proportion of complete transfer events occurring very close to pause sites. The role of pausing in vivo, however, is not clearly understood. By employing a murine leukemia virus-based single-cycle infection assay, strong pausing was shown to increase the probability of recombination, as reflected in the reconstitution of green fluorescent protein expression. The infection assay results were directly correlated with the presence of strong pause sites in reverse transcriptase primer extension assays in vitro. Conversely, when pausing was diminished in vitro, without changing the sequence of the RNA template involved in recombination, there was a significant reduction in recombination in vivo. Together, these data demonstrate that reverse transcriptase pausing, as observed in vitro, directly correlates with recombination during minus-sense DNA synthesis in vivo.

scholarly journals Multiple molecular determinants for retrotransposition in a primer tRNA.

1995 ◽  
Vol 15 (1) ◽  
pp. 217-226 ◽  
Author(s):  
J B Keeney ◽  
K B Chapman ◽  
V Lauermann ◽  
D F Voytas ◽  
S U Aström ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
Specific Interactions ◽  
Acceptor Stem ◽  
Transcription Process ◽  
Molecular Determinants ◽  
Genetic Assay ◽  
Strong Stop ◽  
Interspecies Hybrid

Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.

The Mauriceville plasmid of Neurospora spp. uses novel mechanisms for initiating reverse transcription in vivo

1994 ◽  
Vol 14 (5) ◽  
pp. 3094-3107
Author(s):  
J C Kennell ◽  
H Wang ◽  
A M Lambowitz
Keyword(s):  
Reverse Transcriptase ◽  
Transcription Start Site ◽  
Reverse Transcription ◽  
De Novo ◽  
Minus Strand ◽  
Start Site ◽  
Cdna Synthesis ◽  
Transcription Start

The Mauriceville plasmid and the closely related Varkud plasmid of Neurospora spp. are retroelements that propagate in mitochondria. Replication appears to occur by a novel mechanism in which a monomer-length plasmid transcript having a 3' tRNA-like structure ending in CCA is reverse transcribed to give a full-length minus-strand cDNA beginning at or near the 3' end of the RNA. Here, we show that the plasmids are transcribed in vitro by the Neurospora mitochondrial RNA polymerase, with the major in vitro transcription start site approximately 260 bp upstream of the 5' end of the plasmid transcript. The location of the transcription start site suggests that the monomer-length transcripts are generated by transcription around the plasmid combined with a site-specific RNA cleavage after the 3'-CCA sequence. The 5' ends of minus-strand cDNAs in ribonucleoprotein particles were analyzed to obtain insight into the mechanism of initiation of reverse transcription in vivo. A major class of minus-strand cDNAs begins opposite C2 of the 3'-CCA sequence, the same site used for de novo initiation of cDNA synthesis by the plasmid reverse transcriptase in vitro. A second class of minus-strand cDNAs begins with putative primer sequences that correspond to cDNA copies of the plasmid or mitochondrial transcripts. These findings are consistent with the possibility that the plasmid reverse transcriptase initiates minus-strand cDNA synthesis in vivo both by de novo initiation and by a novel template-switching mechanism in which the 3' OH of a previously synthesized cDNA is used to prime the synthesis of a new minus-strand cDNA directly at the 3' end of the plasmid transcript.

scholarly journals A novel mechanism of self-primed reverse transcription defines a new family of retroelements.

1995 ◽  
Vol 15 (6) ◽  
pp. 3310-3317 ◽  
Author(s):  
H L Levin
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
New Family ◽  
Highly Active ◽  
Priming Process ◽  
Trna Primer ◽  
Compensatory Mutations ◽  
Transcript Evidence

Retroviruses and long terminal repeat (LTR)-containing retrotransposons initiate reverse transcription by using a specific tRNA primer than anneals to the primer-binding site of the retroelement transcript. Sequences from a large number of retroviruses and LTR-containing retrotransposons had indicated that the role of tRNAs in priming reverse transcription is universal among these LTR-containing retroelements. Data presented here strongly support the surprising conclusion that Tf1, a highly active LTR-containing retrotransposon isolated from Schizosaccharomyces pombe, undergoes a novel self-priming process that requires hybridization between the primer-binding site and the first 11 bases of the Tf1 transcript. Single-base mutations in these regions block transposition and reverse transcription, while compensatory mutations that reestablish complementarily rescue both defects. In addition, the sequence of the minus-strand RNA primer of reverse transcription was consistent with its being derived from the 5' end of the Tf1 transcript. Evidence that this mechanism defines a new family of retroelements is presented.

scholarly journals In Vitro Analysis of Human Immunodeficiency Virus Type 1 Minus-Strand Strong-Stop DNA Synthesis and Genomic RNA Processing

2001 ◽  
Vol 75 (2) ◽  
pp. 672-686 ◽  
Author(s):  
Mark D. Driscoll ◽  
Marie-Pierre Golinelli ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Synthesis ◽  
Viral Genome ◽  
Rnase H ◽  
Minus Strand ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Strong Stop

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA3 Lys primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5′ end of the RNA that remained annealed to the 3′ end of the minus-strand strong-stop DNA only if NC was present in the reaction.

scholarly journals The Role of Pr55gag in the Annealing of tRNA3Lys to Human Immunodeficiency Virus Type 1 Genomic RNA

1999 ◽  
Vol 73 (5) ◽  
pp. 4485-4488 ◽  
Author(s):  
Shan Cen ◽  
Yue Huang ◽  
Ahmad Khorchid ◽  
Jean-Luc Darlix ◽  
Mark A. Wainberg ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Primer Binding Site ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA3 Lys, is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA3 Lys in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA3 Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55 gag or Pr160 gag-pol . In vivo placement of tRNA3 Lys is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells with a plasmid coding for either mutant Pr55 gag or mutant Pr160 gag-pol , and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55 gag inhibit tRNA3 Lysplacement. The NC mutations in Pr55 gag reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA3 Lys even in the presence of wild-type Pr55 gag . This dominant phenotype may indicate that the mutant Pr55 gag is disrupting an ordered Pr55 gag structure responsible for the annealing of tRNA3 Lys to genomic RNA.

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