Biochemical characterization of binding of multiple HIV-1 Rev monomeric proteins to the Rev responsive element.
Recombinant HIV-1 Rev protein was overexpressed in Escherichia coli using translational coupling to the beta-glucuronidase gene and demonstrated to interact with high affinity and specificity with the Rev responsive element (RRE). A complex Rev-dependent binding pattern was observed using the gel shift assay which could be simplified to one or two primary bands in the presence of stoichiometric concentrations of RRE. Competition of these bands with a series of homopolymer RNA species demonstrated that Rev is essentially a poly-G binding protein, although poly-I was also shown to compete for specific RRE binding. The stoichiometry of the Rev-dependent gel shift complexes was determined using 125I-labeled Rev. The stable, lowest mobility complex was determined to possess a ratio of between 7 and 8 Rev molecules per RRE containing RNA fragment while the two fastest migrating complexes contained ratios of one and two Rev molecules per RRE, respectively. Using the Hill equation as a model for cooperative interactions, a Hill coefficient of n(app) = 2 was obtained from fitting of direct nitrocellulose filter binding assays, reflecting cooperatively bound Rev molecules on the RRE under equilibrium binding conditions. An increase in ionic strength from 0.0 to 0.3 M NaCl reduced cooperative Rev binding to the RRE, but specificity of Rev for the RRE relative to antisense RNA was increased 100,000-fold. At molar ratios of Rev to RRE above 2, Rev dissociated from the RRE with a T1/2 of approximately 20-25 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Daly
T J
TJ
Repligen Corporation, Cambridge, Massachusetts 02139.
Doten
R C
RC
Rennert
P
P
Auer
M
M
Jaksche
H
H
Donner
A
A
Fisk
G
G
Rusche
J R
JR
D001483:Base Sequence; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics; D015694:Gene Products, rev / Q000378:metabolism*; D015757:Genes, rev; D005966:Glucuronidase / Q000235:genetics; D015497:HIV-1; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011068:Poly G / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D046911:Macromolecular Substances; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D011068:Poly G; D005966:Glucuronidase