Structural Bases of the Annealing of Primer Lys tRNA to the HIV-1 Viral RNA

Tisné, Carine

doi:10.2174/1570162053506919

Cited by 30 publications

(23 citation statements)

References 84 publications

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“…This shows that two distinct parts of tRNA 3 Lys are used by the viral RNA to grip its reverse transcription primer: G183 and G194 respectively contact C72 and C61 on tRNA 3 Lys , at either end of the acceptor stem. This result reconciles apparently conflicting data, ours [6] and those of Hargittai et al [16], which suggested two different initiation sites for annealing, either the centre of the cloverleaf structure or the 3'end unpaired bases of the primer tRNA.…”

Section: -Where Are the Nucleation Sites For Trnalys3/pbs Duplex Formsupporting

confidence: 85%

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New insights into the formation of HIV-1 reverse transcription initiation complex

et al. 2007

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HIV-1 reverse transcriptase uses the host tRNA 3 Lys as a primer for the synthesis of the minus DNA strand. The first event in viral replication is thus the annealing of tRNA to the primer binding site (PBS) in the 5' UTR of the viral RNA. This event requires a major RNA rearrangement which is chaperoned by the viral NC protein. The binding of NC to nucleic acids is essentially non-specific, however, NC is known to bind selectively to hairpins located in the 5' region of the viral RNA. In a previous study, using an NMR approach in which the reaction is slowed down by controlling temperature, we were able to follow details in this RNA unfolding/refolding process and to uncover an intermediate state. We showed that annealing initiates at the junction between the acceptor and the TΨC stems, and that, at physiological temperature, complete annealing is reached only in the presence of NC, probably when the zinc fingers contact the TΨC/D loops.In the present work, we have refined our model of the formation of the tRNA 3 Lys /PBS duplex. First, we show that annealing can initiate both from the single-stranded CCA 3'-end bases of the acceptor stem and from the bases in the TΨC stem. Secondly, by NMR and fluorescence spectroscopy, we have studied the complex between the NC protein and RNA hairpins that mimic the D and T-arms of the tRNA 3 Lys . Interestingly, the NC protein shows strong and specific binding to the D-arm of tRNA 3 Lys , which could explain the overall annealing mechanism.

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Section: -Where Are the Nucleation Sites For Trnalys3/pbs Duplex Formsupporting

confidence: 85%

“…Many of these functions rely on the nucleic acid chaperone activity of NC, i.e. its ability to catalyse nucleic acid conformational rearrangements that lead to the most thermodynamically stable structure (for recent reviews [4][5][6]). …”

Section: Introductionmentioning

confidence: 99%

New insights into the formation of HIV-1 reverse transcription initiation complex

et al. 2007

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“…However, longer RNA transcripts are also used as in the replication of mitochondria DNA and RNA viruses. The reverse-transcriptase of HIV uses host tRNA Lys as the specific primer for the initiation of cDNA synthesis (20). Our current studies show that tRNA can also serve as primers for T7 DNA polymerase.…”

Section: Discussionmentioning

confidence: 72%

“…In the case of HIV, annealing of the tRNA primer to the RNA genome involves a large portion of the tRNA Lys upstream of the CCA 3′ terminus (20). In T7 the ACCA terminus of the tRNA that matches the primase recognition site 5′-TGGT-3′ is sufficient for delivery to T7 DNA polymerase by the primase.…”

Section: Discussionmentioning

confidence: 99%

Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication

Zhu

Lee

Tan

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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DNA primases provide oligoribonucleotides for DNA polymerase to initiate lagging strand synthesis. A deficiency in the primase of bacteriophage T7 to synthesize primers can be overcome by genetic alterations that decrease the expression of T7 gene 5.5, suggesting an alternative mechanism to prime DNA synthesis. The product of gene 5.5 (gp5.5) forms a stable complex with the Escherichia coli histone-like protein H-NS and transfer RNAs (tRNAs). The 3′-terminal sequence (5′-ACCA-3′) of tRNAs is identical to that of a functional primer synthesized by T7 primase. Mutations in T7 that suppress the inability of primase reduce the amount of gp5.5 and thus increase the pool of tRNA to serve as primers. Alterations in T7 gene 3 facilitate tRNA priming by reducing its endonuclease activity that cleaves at the tRNA-DNA junction. The tRNA bound to gp5.5 recruits H-NS. H-NS alone inhibits reactions involved in DNA replication, but the binding to gp5.5-tRNA complex abolishes this inhibition.A 3′-hydroxyl end of an RNA or DNA strand positioned at the catalytic site of DNA polymerase functions as a primer to initiate synthesis of DNA. In most instances, primers are short oligoribonucleotides synthesized by enzymes designated DNA primases (1). DNA primases catalyze the template-directed synthesis of short oligoribonucleotides and stabilize the newly synthesized primer on the template and hand it off to DNA polymerase for initiation of DNA synthesis (2, 3).Bacteriophage T7 has provided a model system for the study of DNA replication. Only three T7 proteins-gene 5 DNA polymerase (gp5), gene 4 primase-helicase (gp4), and gene 2.5 ssDNAbinding protein (gp2.5)-together with host Escherichia coli thioredoxin, are sufficient for leading and lagging strands synthesis in a reconstituted replication system (4). The genes encoding these proteins, together with genes 2 (E. coli RNA polymerase inhibitor), 3 (endonuclease), 3.5 (lysozyme), and 6 (exonuclease) constitute the DNA metabolism class II genes. More than a dozen additional genes within the class II group remain uncharacterized (5).The primase activity of T7 is located in the N-terminal half of the 63-kDa gp4, whereas the C-terminal half of gene 4 encodes the DNA helicase (4). Like other prokaryotic homologs, the primase domain recognizes a specific sequence (1, 6) 5′-(G/T)(G/T) GTC-3′ in the ssDNA template to catalyze the template-directed synthesis of functional tetraribonucleotides (pppACCA, pppACCC, and pppACAC) that the lagging strand DNA polymerase can use as primers to initiate the synthesis of Okazaki fragments. Gene 4 encodes two colinear proteins, the full-length 63-kDa gp4 and a short 56-kDa form in equal amount in vivo from an internal start codon and ribosome-binding site (4). This 56-kDa gp4 has full helicase activity but lacks the N-terminal zinc-binding domain (ZBD), an indispensable motif for primer synthesis (7). Despite its inability to synthesize template-dependent tetraribonucleotides, the 56-kDa gp4 is capable of stabilizing certain preformed oligonucleotides on ...

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“…NC is a versatile protein which is involved in multiple functions during the virus replication cycle, many of these functions relying on the nucleic acid chaperone activity of NC (for recent reviews see Refs. [7,8,9]). In previous studies [10], we have highlighted a strong and specific interaction between the NC protein and the Darm of tRNA Lys 3 .…”

Section: Introductionmentioning

confidence: 99%

Optimizing HSQC experiment for the observation of exchange broadened signals in RNA–protein complexes

Dardel¹,

Tisné²

2007

Comptes Rendus. Chimie

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Sites for interaction in protein-RNA complexes are often regions of conformational exchange. Although the study of exchange processes could bring valuable information about the recognition mode between protein and RNA, chemical exchange can be detrimental to the NMR spectra quality, resulting in broad, very weak or even unobservable signals.In the present report, we used CPMG-like experiments to improve HSQC spectra of an RNA-protein complex in fast exchange on the chemical shift time scale. The use of such improvement will allow us to handle the resolution of the three-dimensional structure of the complex by NMR.

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Structural Bases of the Annealing of Primer Lys tRNA to the HIV-1 Viral RNA

Cited by 30 publications

References 84 publications

New insights into the formation of HIV-1 reverse transcription initiation complex

New insights into the formation of HIV-1 reverse transcription initiation complex

Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication

Optimizing HSQC experiment for the observation of exchange broadened signals in RNA–protein complexes

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