HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

The RNase H activity of reverse transcriptase is essential for retroviral replication. RNA 5-end-directed cleavages represent a form of RNase H activity that is carried out on RNA/DNA hybrids that contain a recessed RNA 5-end. Previously, the distance from the RNA 5-end has been considered the primary determinant for the location of these cleavages. Employing model hybrid substrates and the HIV-1 and Moloney murine leukemia virus reverse transcriptases, we demonstrate that cleavage sites correlate with specific sequences and that the distance from the RNA 5-end determines the extent of cleavage. An alignment of sequences flanking multiple RNA 5-end-directed cleavage sites reveals that both enzymes strongly prefer A or U at the ؉1 position and C or G at the ؊2 position, and additionally for HIV-1, A is disfavored at the ؊4 position. For both enzymes, 5-end-directed cleavages occurred when sites were positioned between the 13th and 20th nucleotides from the RNA 5-end, a distance termed the cleavage window. In examining the importance of accessibility to the RNA 5-end, it was found that the extent of 5-end-directed cleavages observed in substrates containing a free recessed RNA 5-end was most comparable to substrates with a gap of two or three bases between the upstream and downstream RNAs. Together these finding demonstrate that the selection of 5-end-directed cleavage sites by retroviral RNases H results from a combination of nucleotide sequence, permissible distance, and accessibility to the RNA 5-end.In reverse transcription, the single-stranded RNA genome of a retrovirus is transformed into double-stranded DNA by the viral-encoded reverse transcriptase (reviewed in Refs. 1 and 2). This multifunctional enzyme carries out DNA synthesis, strand displacement synthesis, and strand transfer and degrades the RNA portion of RNA/DNA hybrids. The polymerase domain of reverse transcriptase represents the aminoterminal two-thirds of the protein, whereas the RNase H domain comprises the remaining carboxyl-terminal portion. Although the polymerase and RNase H activities are functionally separable, both activities are essential for retroviral replication. The reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) 2 functions as an asymmetric heterodimer composed of p66 and p51 subunits (3), whereas that of Moloney murine leukemia virus (M-MuLV) is a 76-kDa protein that may function as a homodimer or as a monomer (4 -6).RNase H contributes to reverse transcription in three distinct ways (reviewed in Refs. 1, 7, and 8). First, RNase H carries out degradation of the RNA genome both during and after minus-strand DNA synthesis to facilitate plus-strand DNA synthesis and strand transfers. Second, RNase H creates the polypurine tract (PPT) primer from the viral genome. And third, RNase H removes the PPT and tRNA primers used to prime plus-strand and minus-stand DNA synthesis, respectively. Given the vital roles of RNase H in retroviral replication, it is important to understand how RNase H recognizes the RNA/DNA hybri...

Section: Sequence and Distance Are Determinants Of Rna 5ј-end-directedmentioning

confidence: 99%

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confidence: 99%

Sequence, Distance, and Accessibility Are Determinants of 5′-End-directed Cleavages by Retroviral RNases H

Schultz

Zhang

Champoux

2006

“…It is not known if E. coli RNAP or the eucaryotic RNAPs have a single-stranded DNase activity. T7 RNAP nuclease activity resembles the HIV reverse transcriptase nuclease in that arrest of template-specified polymerization is needed (72). The dual activities, viz.…”

Section: Models For Cleavage and Unusual Polymerization During Elongamentioning

confidence: 99%

Nuclease Activity of T7 RNA Polymerase and the Heterogeneity of Transcription Elongation Complexes

Sastry

Ross

1997

We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono-or dinucleotide steps. The nuclease is a 3 to 5 exonuclease leaving a 3 -OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg 2؉ . The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3 -end-initiated polymerase activity is unmasked. RNA from isolated preelongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3 -end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3 -end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.Transcription by procaryotic DNA-dependent RNA polymerases can be represented as follows (Scheme I).Elongation Termination SCHEME I. Representation of a transcription cycle. Open complexes synthesize short RNAs (up to 10 nts) during abortive initiation. After clearing the promoter, RNAP enters the elongation phase. Termination occurs at certain DNA sequences in either a factor-dependent or a factor-independent manner (2). The current view of transcription elongation has been possible because of our ability to arrest elongation at specific sites on DNA templates, partial purification of arrested complexes, and the enzymatic and chemical probing of their structures (3-17).The unexpected discovery of an RNA cleavage reaction in arrested Escherichia coli complexes (18) was followed by documentation of analogous cleavage reactions in other RNAPs (19 -23). E. coli RNAP and eucaryotic RNAP II are capable of RNA cleavage in binary and ternary complexes (24 -26). GreA and GreB of E. coli enhance the intrinsic cleavage by E. coli RNAP (25,27,28). GreA and GreB (and a eucaryotic counterpart, SII) prevent elongation arrest (29). GreA may participate in the fidelity of RNA synthesis (30).T7 RNAP (98.8 kDa) belongs to a class of single-subunit RNAPs that includes T3 and SP6 phage RNAPs (31-33). The three-dimensional structure of T7 RNAP shows high ␣-helicity with a deep cleft. Using a novel photochemical cross-linking technique, we have id...

“…RNase H(Ϫ) HIV-1 RT bearing the E478Q mutation was purified essentially as described previously (32). This mutant polymerase was used to prevent cleavage of the RNA template by the double-stranded RNase activity associated with HIV-1 RNase H (33). The wild-type and mutant enzymes are equally efficient in initiating reverse transcription (18).…”

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confidence: 99%

Dynamics of the HIV-1 Reverse Transcription Complex during Initiation of DNA Synthesis

Lanchy

Isel

Keith

et al. 2000

Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA 3 Lys and reverse transcriptase (RT). The vRNA and the primer tRNA 3 Lys form several intermolecular interactions in addition to annealing of the primer 3 end to the primer binding site (PBS). These interactions are crucial for the efficiency and the specificity of the initiation of reverse transcription. However, as they are located upstream of the PBS, they must unwind as DNA synthesis proceeds. Here, the dynamics of the complex during initiation of reverse transcription was followed by enzymatic probing. Our data revealed reciprocal effects of the tertiary structure of the vRNA⅐tRNA 3 Lys complex and reverse transcriptase (RT) at a distance from the polymerization site. The structure of the initiation complex allowed RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modified the tertiary structure of the complex at 10 -14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA sequences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial complex, indicating that the fate of the tRNA depended on the tertiary structure of the initial complex.Reverse transcription is a key step in the retroviral replication cycle (1, 2), during which the virus-encoded reverse transcriptase (RT), 1 which possesses RNA-and DNA-dependent DNA polymerase and RNase H activities (3), converts the genomic RNA into double-stranded DNA. In retroviruses, initiation of reverse transcription requires annealing of the 18 3Ј-terminal nucleotides of a cellular tRNA to the primer binding site (PBS), located near the 5Ј end of the RNA genome (4 -6).In human immunodeficiency virus type 1(HIV-1), a strong selective pressure maintains tRNA 3 Lys as primer. Mutating the PBS to match the 3Ј end of other tRNAs dramatically reduces viral replication, and rapid reversion to the wild-type PBS is observed (7-9). A similar situation also prevails in avian leukosis viruses (10). In vitro, HIV-1 RT is able to discriminate against non-self-tRNA primers (7, 11). Interestingly, the specificity of the initiation of HIV-1 reverse transcription correlates with virus-specific interactions between the primer tRNA 3 Lys and the viral RNA (vRNA). Chemical and enzymatic probing revealed intricate intermolecular interactions between the anticodon loop and stem, part of the variable loop of tRNA 3Lys , and sequences upstream of the PBS (12, 13) (Fig. 1). Extended tRNA-vRNA interactions were also identified in avian retroviruses (14, 15) and in yeast retrotransposon Ty1 (16) and proposed in HIV-2 (17).In HIV-1, the best-characterized specific intermolecular interaction involves the anticodon loop of tRNA 3Lys and an A-rich...