Sequence Specific Thermodynamic and Structural Properties for DNA.cntdot.RNA Duplexes

Ratmeyer, L.; Vinayak, Ravi; Zhong, Yi Yi; Zon, G.; Wilson, W. David

doi:10.1021/bi00183a037

Cited by 175 publications

(173 citation statements)

References 22 publications

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“…2A for each extremity of the PPT (LNA 15/14 and LNA 2/1) as well as the portion of the PPT where unusual base-pairing leads to unstacking of template base Ϫ11C (LNA 11/10) (4, 45). As might be expected for an RNA/DNA hybrid (7,54,55), the spectrum of an unmodified duplex displays a positive band at 267 nm, with minima at 245 and 210 nm. Introducing adjacent LNA analogs at positions Ϫ15/Ϫ14, Ϫ11/Ϫ10, or Ϫ2/Ϫ1 induces a minor change in the 210-nm minimum, suggesting local A-like geometry.…”

Section: Resultssupporting

confidence: 66%

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Dash

Yi-Brunozzi

Grice

2004

Journal of Biological Chemistry

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Unusual base-pairing in a co-crystal of reverse transcriptase (RT) and a human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/ DNA hybrid suggests local nucleic acid flexibility mediates selection of the plus-strand primer. Structural elements of HIV-1 RT potentially participating in recognition of this duplex include the thumb subdomain and the ribonuclease H (RNase H) primer grip, the latter comprising elements of the connection subdomain and RNase H domain. To investigate how stabilizing HIV-1 PPT structure influences its recognition, we modified the (؊) DNA template by inserting overlapping locked nucleic acid (LNA) doublets and triplets. Modified RNA/ DNA hybrids were evaluated for cleavage at the PPT/U3 junction. Altered specificity was observed when the homopolymeric dA⅐rU tract immediately 5 of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT⅐rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC⅐rG and neighboring dT⅐rA tract were minimally affected by LNA insertion, RNase H activity was highly sensitive to altering the junction between these structural elements. Using 3 -end-labeled PPT RNA primers, we also identified novel cleavage sites ahead (؉5/؉6) of the PPT/U3 junction. Differential cleavage at the PPT/U3 junction and U3 ؉ 5/؉6 site in response to LNA-induced template modification suggests two binding modes for HIV-1 RT, both of which may be controlled by the interaction of its thumb subdomain (potentially via the minor groove binding track) at either site of the unzipped region.Minus (Ϫ)-strand DNA synthesis in retroviruses is accompanied by degradation of viral RNA of the RNA/DNA replication intermediate. These are events mediated by the N-terminal DNA polymerase and C-terminal ribonuclease H (RNase H) 1 catalytic centers of the multifunctional reverse transcriptase (RT), respectively (1). Correct positioning of nucleic acids to facilitate each of these steps clearly requires its specific interaction with structural motifs of the virus-coded polymerase. Crystallographic (2-4) and biochemical analysis (5-13) of human immunodeficiency virus type 1 (HIV-1) RT identified the primer grip of the p66 thumb as a motif critical for positioning the primer 3ЈOH before nucleophilic attack on the incoming dNTP. Immediately adjacent to this motif, the minor groove binding track (MGBT) or translocation track is proposed to mediate translocation of the polymerization machinery and act as a sensor of nucleic acid geometry (14 -19). Recently, it was proposed that the RNase H primer grip (RHPG), involving portions of the p66 connection subdomain and RNase H domain, interacts with the DNA strand of an RNA/DNA hybrid, providing the RNA with a trajectory appropriate for hydrolysis within the RNase H catalytic center (4, 20, 21). These combined activities effectively prepare nascent (Ϫ) DNA for use as template for plus (ϩ)-strand, DNA-dependent DNA synthesis.A critical...

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Section: Resultssupporting

confidence: 66%

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Dash

Yi-Brunozzi

Grice

2004

Journal of Biological Chemistry

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“…As expected, the all-RNA duplex was more stable than the all-DNA duplex (66,70,71), whereas the chimeras were intermediate in their thermal stability. The relative stability of chimeric DNA-RNA duplexes is difficult to predict and depends strongly on base composition (65-67, 70, 71).…”

Section: Melting Studies Of Duplexes Between the Pbs (Template) And Vsupporting

confidence: 76%

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro

Iwatani¹,

Rosen²,

Guo³

et al. 2003

Journal of Biological Chemistry

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“…Newly initiated transcripts of up to 6 nucleotides formed on SUP4-CTC2 are expected to exist as a rPy:dPu heteroduplex. Heteroduplexes of this type are less stable than, and structurally distinct from, either the corresponding rPu:dPy heteroduplex or the rPu:rPy and dPu:dPy homoduplexes (7,16,27,38,40). However, there is some question as to whether these concepts of heteroduplex stability and structure arising from studies of long polynucleotides can be directly applied to the short nascent RNA in a newly formed transcription complex.…”

Section: Discussionmentioning

confidence: 99%

Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

Zecherle

Whelen

Hall

1996

Molecular and Cellular Biology

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We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene. The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1. Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding the ؉1 and ؉2 nucleotides of pre-tRNA Tyr , had a strong deleterious effect in vivo, as did the more extensive change CTCCTC. In contrast, mutant genes bearing each of the possible single changes at nucleotide ؉1 retained normal suppression levels. The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site. ATP was preferentially utilized as the 5 nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the ؉1 nucleotide. Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex. Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o. Our results suggest that the purine-rich sequences at the 5 ends of the nascent transcripts of many genes act to stabilize the early ternary complex.The faithful and efficient transcription of DNA into RNA is a primary regulatory point in gene expression. The relative rates at which different genes are transcribed by the same RNA polymerase are strongly influenced by sequences surrounding the transcription start site. Previous studies of these contextual effects have emphasized sequence differences in the formation of the initiation complex and in the selection of the initiation site. RNA polymerases, both bacterial and eukaryotic, preferentially initiate transcription with a purine residue (4,5,8,14,18,29,30). In contrast, pyrimidine-specific transcription start sites are far less frequent as judged by 5Ј-end determination of RNAs synthesized both in vivo and in vitro (5, 18); only 7% of 88 Escherichia coli promoters surveyed by Hawley and McClure (14) directed transcription start sites with either UTP or CTP, with pyrimidine-specific initiation occurring only in the absence of purines within a defined window (36). The specific binding of purines at the i site of E. coli RNA polymerase, both in the presence and in the absence of template DNA, suggests that the interaction of purine ribonucleotides with RNA polymerase is important for productive transcription initiation (1, 47-49). However, the precise role in this process of nucleic acid sequences at and immediately following position ϩ1 remains unclear.As a system for studying small or large quantitative effects upon the level of transcription of a gene, RNA polymerase III transcription of the SUP4 gene ...

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Sequence Specific Thermodynamic and Structural Properties for DNA.cntdot.RNA Duplexes

Cited by 175 publications

References 22 publications

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro

Purines Are Required at the 5′ Ends of Newly Initiated RNAs for Optimal RNA Polymerase III Gene Expression

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