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D W Rodgers

Publications and source records attributed to D W Rodgers.

12 recordsLinked to original sources
PubMed1996

Alterations to the primer grip of p66 HIV-1 reverse transcriptase and their consequences for template-primer utilization.

Alanine scanning mutagenesis was undertaken to evaluate the structural significance of Met230-His235 of the 66 kDa subunit of p66/p51 human immunodeficiency virus reverse transcriptase (HIV-1 RT). Together with Glu224-Trp229, these residues provide the framework of the p66 "primer grip", whose proposed role is maintaining the primer terminus in an orientation appropriate for nucleophilic attack on an incoming dNTP. Of these residues, altering Leu234 results in a p66 subunit incapable of associating into heterodimer. The remaining selectively mutated enzymes were successfully reconstituted and purified to homogeneity for evaluation of RT-associated activities. We show here that alterations to any residue within the p66-Trp229-Met230-Gly231-Tyr232-quartet alter functions associated with both the DNA polymerase and ribonuclease H (RNase H) domains. Detailed analysis of mutant p66Y232A/p51 with an intact or a model "precleaved" RNA-DNA hybrid suggests an altered RNase H phenotype could result from relocation of template-primer in the nucleic acid binding cleft. As a consequence, template nucleotide-8 is positioned in the immediate vicinity of the RNase H catalytic center rather than nucleotide-17.

Amino Acid Sequence↗
PubMed1995

The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1.

The crystal structure of the reverse transcriptase (RT) from the type 1 human immunodeficiency virus has been determined at 3.2-A resolution. Comparison with complexes between RT and the polymerase inhibitor Nevirapine [Kohlstaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A. & Steitz, T.A. (1992) Science 256, 1783-1790] and between RT and an oligonucleotide [Jacobo-Molina, A., Ding, J., Nanni, R., Clark, A. D., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H. & Arnold, E. (1993) Proc. Natl. Acad. Sci. USA 90, 6320-6324] reveals changes associated with ligand binding. The enzyme is a heterodimer (p66/p51), with domains labeled "fingers," "thumb," "palm," and "connection" in both subunits, and a ribonuclease H domain in the larger subunit only. The most striking difference between RT and both complex structures is the change in orientation of the p66 thumb (approximately 33 degrees rotation). Smaller shifts relative to the core of the molecule were also found in other domains, including the p66 fingers and palm, which contain the polymerase active site. Within the polymerase catalytic region itself, there are no rearrangements between RT and the RT/DNA complex. In RT/Nevirapine, the drug binds in the p66 palm near the polymerase active site, a region that is well-packed hydrophobic core in the unliganded enzyme. Room for the drug is provided by movement of a small beta-sheet within the palm domain of the Nevirapine complex. The rearrangement within the palm and thumb, as well as domain shifts relative to the enzyme core, may prevent correct placement of the oligonucleotide substrate when the drug is bound.

Binding Sites↗
PubMed1994

Cryocrystallography.

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Crystallography, X-Ray↗
PubMed1993

The complex between phage 434 repressor DNA-binding domain and operator site OR3: structural differences between consensus and non-consensus half-sites.

BACKGROUND: The repressor of phage 434 binds to a set of operator sites as a homodimer. Its relative affinities for these sites determine the switch from lysogenic to lytic growth. The six 434 operator sites (OR1, OR2, OR3, OL1, OL2 and OL3) have a particularly simple organization; all are 14 base pairs long, with a conserved 5'-ACAA sequence symmetrically placed at either end, and a variable central six base pairs. OR3 is unique among naturally-occurring 434 operator sites in that it contains a non-consensus base pair, G.C, at the fourth position of the otherwise invariant 5'-ACAA sequence. Comparisons among structures of the 434 repressor DNA-binding domain, R1-69, bound to various operator sites, allow us to analyze differential specificity in regulatory complexes of this kind. RESULTS: We have determined the structure at 2.5 A resolution of a complex of R1-69 with DNA containing the OR3 site and compared it with previously studied complexes of R1-69 bound to OR1 and OR2. There are surprisingly extensive structural differences between the consensus and non-consensus half-sites of OR3 with respect to their interactions with R1-69, including a shift in the DNA backbone and a small rotation of the entire R1-69 monomer. CONCLUSIONS: Recognition of the base pair difference that is critical for the 434 regulatory switch involves a number of amino acid residues, not just the one or two side chains in direct contact with the G-C base pair. Moreover, the repressor imposes a somewhat altered DNA conformation on the non-consensus half-site.

Amino Acid Sequence↗
PubMed1992

Tritium dynamics in mice exposed to tritiated water and diet.

The contributions of tritiated water (3HHO) and dietary tritiated amino acids to the steady-state specific activities of tissue water tritium and organically bound tritium in mice were essentially independent and additive. Following exposure (56 d), organically bound tritium clearance was resolved into two distinct compartments. The first, with a half-life of 1-2 d, likely represented exchangeable organically bound tritium, and the second, with a half-life of 20-30 d, likely represented nonexchangeable organically bound tritium. Since organically bound tritium was cleared much more slowly than tissue water tritium, organically bound tritium was the principal determinant in estimated radiation doses to mice following exposure.

Amino Acids↗
PubMed1990

Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains.

The structure of an N-terminal fragment of CD4 has been determined to 2.4 A resolution. It has two tightly abutting domains connected by a continuous beta strand. Both have the immunoglobulin fold, but domain 2 has a truncated beta barrel and a non-standard disulphide bond. The binding sites for monoclonal antibodies, class II major histocompatibility complex molecules, and human immunodeficiency virus gp120 can be mapped on the molecular surface.

Amino Acid Sequence↗
PubMed1988

Recognition of a DNA operator by the repressor of phage 434: a view at high resolution.

The repressors of temperate bacteriophages such as 434 and lambda control transcription by binding to a set of DNA operator sites. The different affinity of repressor for each of these sites ensures efficient regulation. High-resolution x-ray crystallography was used to study the DNA-binding domain of phage 434 repressor in complex with a synthetic DNA operator. The structure shows recognition of the operator by direct interactions with base pairs in the major groove, combined with the sequence-dependent ability of DNA to adopt the required conformation on binding repressor. In particular, a network of three-centered bifurcated hydrogen bonds among base pairs in the operator helps explain why 434 repressor prefers certain sites over others. These bonds, which stabilize the conformation of the bound DNA, can form only with certain sequences.

Base Composition↗
PubMed1987

Pairings and polarities of the 14 strands in sickle cell hemoglobin fibers.

Sickle cell anemia results from the formation of hemoglobin S fibers in erythrocytes, and a greater understanding of the structure of these fibers should provide insights into the basis of the disease and aid in the development of effective antisickling agents. Improved reconstructions from electron micrographs of negatively stained single hemoglobin S fibers or embedded fiber bundles reveal that the 14 strands of the fiber are organized into pairs. The strands in each of the seven pairs are half-staggered, and from longitudinal views the polarity of each pair can be determined. The positions of the pairs and their polarities (three in one orientation; four in the opposite orientation) suggest a close relationship with the crystals of deoxyhemoglobin S composed of antiparallel pairs of half-staggered strands.

Crystallization↗
PubMed1986

Tritium dynamics in juvenile rainbow trout, Salmo gairdneri.

Juvenile rainbow trout, Salmo gairdneri, were maintained in tritiated water, fed a tritiated diet or both, and the specific activity of 3H in the fish was determined during and following 3H exposure. Tissue water 3H-equilibrated rapidly with the ambient water (estimated half-life less than 30 min) and maintained a steady-state specific activity similar to ambient. In contrast, the steady-state specific activity of organically bound 3H was significantly affected by that of the diet. The steady-state specific activity of organically bound 3H of trout maintained in tritiated water but fed non-tritiated diets was approximately 20% of that of the ambient water. This steady-state specific activity was achieved relatively quickly (half-life approximately 1 d) and presumably reflects the exchangeable H of the fish. For trout fed a tritiated diet, in either tritiated or non-tritiated water, steady-state specific activities of organically bound 3H were on the order of 80% of that of the diet. These steady-state specific activities were reached much more slowly (estimated half-lives of 18-32 d) and are likely to represent the accumulation of non-exchangeable 3H in the organic material of the trout.

Animals↗
PubMed1984

The polymerization of nickel (II) hemoglobin S under aerobic conditions.

Sickle hemoglobin (Fe(II)HbS) reconstituted with nickel(II)protoporphyrin IX yields an artificial hemoglobin (Ni(II)HbS), the first heme-substituted hemoglobin shown to mimic the polymerization of deoxyHbS. Unlike Fe(II)HbS, Ni(II)HbS does not bind oxygen and therefore polymerizes under aerobic conditions. While the polymer solubility coefficient (Csat) for Ni(II)HbS is elevated about 2 g/100 mL compared with that for deoxy Fe(II)HbS, hemoglobin concentration in the polymer phase is the same. Electron micrographs of thin sections of embedded Ni(II)HbS reveal 20-nm-diameter fibers indistinguishable from those seen with deoxygenated native HbS. Nickel(II)HbS can be used in studies on the sickling process and on antisickling agents that could not previously be done or were difficult to execute because of the need for an anaerobic environment.

Aerobiosis↗