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)

The lB-ketoadipate pathway of Acinetobacter caloaceticus comprises two parallel metabolic branches. One branch, mediated by six enzymes encoded by the cat genes, converts catechol to succinate and acetyl coenzyme A (acetyl-CoA); the other branch, catalyzed by products of the pca genes, converts protocatechuate to succinate and acetyl-CoA by six metabolic reactions analogous or identical to those of the catechol sequence. We used the expression plasmid pUC18 to construct expression libraries of DNA from an A. caloaceticus mutant strain from which the cat genes had been deleted. Immunological screening with antiserum to the pcaE gene product, ,B-ketoadipate:succinyl-CoA transferase I, resulted in the isolation of a cloned 11-kilobase-pair (kbp) fragment which inducibly expressed all six pea genes under control of the lac promoter on pUC18. The induced Escherichia coli cells formed the six pea gene products at levels 10-to 30-fold higher than found in fully induced A. calcoaceicus cultures, although protocatechuate 3,4-dioxygenase (the iron-containing product of the pcaA gene) from the recombinant strain possessed a relatively low turnover number. An E. coli culture expressing the cloned pea genes quantitatively converted protocatechuate to (-ketoadipate; failure of the organism to metabolize the latter compound can be most readily ascribed to relatively low pool levels of succinyl-CoA, a required substrate for 0-ketoadipate:succinyl-CoA transferase, in E. coli. The gene order and direction of transcription were determined to be pcACBDFE by identification of enzymes expressed in subclones, by using natural transformation to identify subclones carrying DNA corresponding to dysfunctional alleles in mutant A. calcoaceticus strains, and by restriction mapping of both the ll-kbp fragment and derivatives of the ll-kbp fragment containing TnS in the pcaA, pcaB, peaC, pcaD, and peaE genes. The fragment containing the pea gene hybridized strongly and specifically to a previously cloned fragment containing A. caloaceticus cat genes.The pca structural genes encode six enzymes that convert protocatechuate to citric acid cycle intermediates via Pketoadipate ( Fig. 1; 27). Protocatechuate induces coordinate synthesis of the enzymes in Acinetobacter calcoaceticus (3), and their unified transcriptional control has been suggested by their constitutive formation in regulatory mutants (4,22). The physical organization of the A. calcoaceticus pca genes has not been explored. The A. calcoaceticus cat genes encode six enzymes that convert catechol to citric acid cycle intermediates by reactions analogous or identical to those catalyzed by the pca gene products (Fig. 1) To establish a basis for analysis of possible interaction between cat and pca genes in A. calcoaceticus, we constructed expression libraries of A. calcoaceticus DNA and used immunological screening to identify a clone that expressed the pcaE gene. The cloned DNA was an 11-kbp EcoRI fragment that hybridized with the 5-kbp EcoRI fragment containing the catBCDE genes and pos...

Perturbations within the transactivation and carboxy-terminal domains of HIV-1 Rev were examined for effects on Rev responsive element (RRE) binding activities in vitro and biological activity in vivo. Binding affinities, specificities, and multimerization of the transactivation mutants M10 and Rev/Rex M10-16 on the RRE were equivalent to wild-type Rev. Substitution of the Rex transactivation domain within Rev resulted in the incorporation of an internal methionine residue which, when cleaved with CNBr and subsequently purified, produced a protein species (CNBr-Rev) unable to fully multimerize on the RRE. Instead, two discrete protein-dependent species were generated in the gel shift assay. Furthermore, CNBr-Rev was observed to bind to the RRE with high specificity and an equilibrium binding constant of 6 x 10(-10) M. A C-terminal Rev deletion mutant (Rev M9 delta 14) lacking amino acids 68-112 displayed identical RRE binding characteristics to the CNBr-Rev protein. This protein, which lacks both the activation and the C-terminal domains, was biologically inactive but maintained the ability to discriminate the RRE from nonspecific RNA. Deletion of amino acids 92-112 resulted in a Rev mutant (Rev M11 delta 14) which bound to the RRE with wild-type affinity and high specificity. This purified mutant was observed to be aberrant in multimerization activity on the RRE with reduced multimerization apparent in the gel shift assay. However, Rev M11 delta 14 possessed biological activity equivalent to wild-type Rev in a cell-based p24 ELISA assay. These results suggest that polymerization on the RRE is dispensable for Rev activity and that two monomeric Rev proteins bound to the RRE are sufficient for biological activity. Furthermore, in vivo experiments using the Rev/Rex chimeric mutant and the M10 transdominant mutant as well as in vitro dissociation rate studies with Rev M11 delta 14 and Rev M9 delta 14 suggest that the M9 through M11 domain of the protein may be involved in RRE-dependent specific Rev dimerization.

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