Ribonuclease H activity during initiation of reverse transcription using tRNA(lys)/RNA primer/template of human immunodeficiency virus. 1996

H B Artzi, and J Shemesh, and E Zeelon, and B Amit, and L Kleiman, and M Gorecki, and A Panet
Bio-Technology General Israel, Ltd., Kiryat Weizmann, Rehovot, Israel.

The specificity of the initial cleavage by the RNaseH activity of HIV-1 reverse transcriptase (RT) during minus strong-stop DNA synthesis was studied using the authentic primer/template tRNA(lys)/HIV RNA. We observed that concomitant with the initiation of DNA synthesis, RNaseH activity of HIV RT introduced the first endonucleolytic cuts within the U5 region of the HIV RNA template, mainly 1 and 3 bases away from the primer binding site. To analyze whether the cleavage sites were determined by sequence specificity, the authentic U5 region at one of the cleavage sites was mutated. The change of sequence did not alter the initial cleavage pattern of RNaseH. In order to determine the size of the RNA/DNA hybrid that is required for RNaseH activation during reverse transcription initiation, DNA synthesis was limited by dideoxynucleotides. DNA extension of the tRNA(lys) primer by 17 deoxyribonucleotides but not by 6 deoxyribonucleotides was sufficient to activate the RNaseH site of HIV RT. Taken together, our results indicate that during initiation of minus strongstop DNA synthesis by HIV RT, the first RNaseH-mediated endonucleolytic cut of the genomic RNA is dictated mainly by the length of the nascent DNA and not by sequence preference.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D004789 Enzyme Activation Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme. Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D012194 RNA-Directed DNA Polymerase An enzyme that synthesizes DNA on an RNA template. It is encoded by the pol gene of retroviruses and by certain retrovirus-like elements. EC 2.7.7.49. DNA Polymerase, RNA-Directed,RNA-Dependent DNA Polymerase,Reverse Transcriptase,RNA Transcriptase,Revertase,DNA Polymerase, RNA Directed,DNA Polymerase, RNA-Dependent,RNA Dependent DNA Polymerase,RNA Directed DNA Polymerase
D012313 RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed) RNA, Non-Polyadenylated,Ribonucleic Acid,Gene Products, RNA,Non-Polyadenylated RNA,Acid, Ribonucleic,Non Polyadenylated RNA,RNA Gene Products,RNA, Non Polyadenylated
D012357 RNA, Transfer, Lys A transfer RNA which is specific for carrying lysine to sites on the ribosomes in preparation for protein synthesis. Lysine-Specific tRNA,Transfer RNA, Lys,tRNALys,tRNA(Lys),Lys Transfer RNA,Lysine Specific tRNA,RNA, Lys Transfer,tRNA, Lysine-Specific
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA

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