HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.
- MeSH
- DNA primery biosyntéza chemie MeSH
- DNA virů MeSH
- HIV reverzní transkriptasa metabolismus fyziologie MeSH
- HIV-1 genetika MeSH
- konformace nukleové kyseliny MeSH
- krystalografie rentgenová metody MeSH
- nukleové kyseliny MeSH
- poly A MeSH
- poly U MeSH
- polynukleotidy MeSH
- puriny chemie MeSH
- ribonukleasa H metabolismus MeSH
- RNA virová chemie MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.
- MeSH
- aminoacylace tRNA MeSH
- fosforylace MeSH
- peptidyltransferasy metabolismus MeSH
- podjednotky ribozomu metabolismus MeSH
- poly U genetika metabolismus MeSH
- ribozomální proteiny metabolismus MeSH
- Streptomyces coelicolor enzymologie genetika metabolismus MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A complete series of the 2 '-5 ' and 3 '-5 ' regioisomeric types of r(ApA) and 2 '-d(ApA) analogues with the α-hydroxy-phosphonate C3 '-O-P-CH(OH)-C4 ″ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 '-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 '-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 '(2 ')-O-P-CH(2)-C4 ″ linkage lacking the 5 '-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 '-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.
- MeSH
- antivirové látky terapeutické užití MeSH
- bakteriální polysacharidy terapeutické užití MeSH
- fluorescenční protilátková technika MeSH
- gama-globuliny terapeutické užití MeSH
- induktory interferonů terapeutické užití MeSH
- keratitida dendritická farmakoterapie imunologie mikrobiologie prevence a kontrola MeSH
- konjunktiva imunologie mikrobiologie MeSH
- králíci MeSH
- lipopolysacharidy normy MeSH
- modely nemocí na zvířatech MeSH
- neutralizační testy MeSH
- placebo MeSH
- poly A-U terapeutické užití MeSH
- prednisolon terapeutické užití MeSH
- Salmonella typhi MeSH
- Simplexvirus imunologie izolace a purifikace MeSH
- tvorba protilátek MeSH
- virus Semliki forest MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
