BIOMAT Database Logo BIOMAT Database Logo

Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. 1995

P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854-5638, USA.

When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
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
D009690 Nucleic Acid Conformation The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape. DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D011487 Protein Conformation The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain). Conformation, Protein,Conformations, Protein,Protein Conformations
D004261 DNA Replication The process by which a DNA molecule is duplicated. Autonomous Replication,Replication, Autonomous,Autonomous Replications,DNA Replications,Replication, DNA,Replications, Autonomous,Replications, DNA
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
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

Related Publications

P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Structure and function of HIV-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition.
January 2009, Journal of molecular biology,
P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Molecular insights into the mechanisms of HIV-1 reverse transcriptase resistance to nucleoside analogs.
May 2006, Mini reviews in medicinal chemistry,
P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Stepping statistics of single HIV-1 reverse transcriptase molecules during DNA polymerization.
August 2005, The journal of physical chemistry. B,
P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
HIV reverse transcriptase structure-function relationships.
July 1991, Biochemistry,
P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure.
April 1990, Virology,
P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Structure-function studies of HIV reverse transcriptase.
January 1990, Annals of the New York Academy of Sciences,
P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Single-molecule study of DNA polymerization activity of HIV-1 reverse transcriptase on DNA templates.
February 2010, Journal of molecular biology,
P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase.
November 2015, Acta crystallographica. Section F, Structural biology communications,
P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Structural and kinetic insights into HIV-1 reverse transcriptase inhibition by farnesiferol C.
March 2021, International journal of biological macromolecules,
P H Patel, and A Jacobo-Molina, and J Ding, and C Tantillo, and A D Clark, and R Raag, and R G Nanni, and S H Hughes, and E Arnold
Evolving understanding of HIV-1 reverse transcriptase structure, function, inhibition, and resistance.
April 2020, Current opinion in structural biology,
Copied contents to your clipboard!