Biomedical subjects

F W Whipple

Publications and source records attributed to F W Whipple.

5 recordsLinked to original sources

PubMed1998

Amino acid-amino acid contacts at the cooperativity interface of the bacteriophage lambda and P22 repressors.

The bacteriophage lambda repressor and its relatives bind cooperatively to adjacent as well as artificially separated operator sites. This cooperativity is mediated by a protein-protein interaction between the DNA-bound dimers. Here we use a genetic approach to identify two pairs of amino acids that interact at the dimer-dimer interface. One of these pairs is nonconserved in the aligned sequences of the lambda and P22 repressors; we show that a lambda repressor variant bearing the P22 residues at these two positions interacts specifically with the P22 repressor. The other pair consists of a conserved ion pair; we reverse the charges at these two positions and demonstrate that, whereas the individual substitutions abolish the interaction of the DNA-bound dimers, these changes in combination restore the interaction of both lambdacI and P22c2 dimers.

Amino Acid Sequence↗

PubMed1998

Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli.

This report describes an Escherichia coli genetic system that permits bacterial genetic methods to be applied to the study of essentially any prokaryotic or eukaryotic site-specific DNA binding protein. It consists of two parts. The first part is a set of tools that facilitate construction of customized E.coli strains bearing single copy lacZYA reporters that are repressed by a specific target protein. The second part is a pair of regulatable protein expression vectors that permit in vivo production of the target protein at levels appropriate for genetic experiments. When expressed in a properly designed reporter strain, the target protein represses the lac genes, resulting in an E.coli phenotype that can be quantitatively measured or exploited in large scale genetic screens or selections. As a result, large plasmid-based libraries of protein genes or pools of mutagenized variants of a given gene may be examined in relatively simple genetic experiments. The strain construction technique is also useful for generating E.coli strains bearing reporters for other types of genetic systems, including repression-based and activation-based systems in which chimeric proteins are used to examine interactions between foreign protein domains.

Base Sequence↗

PubMed1997

The activation defect of a lambda cI positive control mutant.

"Positive control" mutants of the cI protein of bacteriophage lambda (lambda cI) bind DNA but, unlike the wild-type protein, fail to activate transcription. According to the original interpretation of Ptashne and co-workers, these mutants bear amino acid substitutions that disrupt a stimulatory interaction between lambda cI bound at operator site O(R)2 and RNA polymerase bound at promoter P(RM), an idea supported by kinetic analysis in one case. Genetic analysis has suggested that one residue in particular, glutamate 34 (E34), is critical for the stimulatory effect of wild-type lambda cI. More recently, however, Kolkhof and Muller-Hill have challenged this view, suggesting that mutant E34K fails to activate because it binds at unusually low concentrations to O(R)3, a site that mediates repression of P(RM). To test this hypothesis, we have examined the behaviour of the lambda cI-E34K mutant both in vitro and in vivo by assaying transcription from P(RM) and monitoring operator site occupancy over a range of protein concentrations. Our results are inconsistent with the interpretation of Kolkhof and Muller-Hill, and demonstrate that under conditions where lambda operator O(R)2 is fully occupied and operator O(R)3 is vacant, wild-type lambda cI activates transcription from promoter P(RM) whereas the mutant does not.

Bacteriophage lambda↗

PubMed1994

Specificity determinants for the interaction of lambda repressor and P22 repressor dimers.

The related phage lambda and phage P22 repressors each bind cooperatively to adjacent and separated operator sites, an interaction that involves a pair of repressor dimers. The specificities of these interactions differ: Each dimer interacts with its own type but not with dimers of the heterologous repressor. The two repressors exhibit significant amino acid sequence homology in their carboxy-terminal domains, which are responsible for both dimer formation and the dimer-dimer interaction. Here, we identify a collection of amino acid substitutions that disrupt the protein-protein interaction of DNA-bound lambda repressor dimers and show that several of these substitutions have the same effect when introduced at the corresponding positions of P22 repressor. We use this information to construct a variant of the lambda repressor bearing only six non-wild-type amino acids that has a switched specificity; that is, it binds cooperatively with P22 repressor, but not with wild-type lambda repressor. These results identify a series of residues that determine the specificities of the two interactions.

Amino Acid Sequence↗

PubMed1992

Mechanism of initiation of transcription by Bacillus subtilis RNA polymerase at several promoters.

The behavior of the major vegetative cell RNA polymerase of Bacillus subtilis, E sigma A, during initiation of transcription was compared to that of its Escherichia coli counterpart, E sigma 70, at several promoters known to be actively transcribed by both RNA polymerases. Challenge experiments using heparin, restriction endonucleases, and competing promoter DNA under various conditions showed that, at several promoters, complexes with B. subtilis RNA polymerase formed in the absence of nucleoside triphosphates were unstable. These complexes produced DNase I footprints that were less extended than those produced by the E. coli enzyme at the same promoters. Further, in the presence of certain combinations of nucleoside triphosphates, conditions that allow production of abortive oligonucleotides, these B. subtilis RNA polymerase complexes remained dissociable. Thus, at these promoters, the B. subtilis enzyme interacted with the DNA and reached a catalytically active initial transcribing complex without becoming committed to the template. At these same promoters, E. coli RNA polymerase formed stable open complexes before forming any phosphodiester bonds. B. subtilis initial transcribing complexes also remained sensitive to the drug rifampicin until a later stage in the initiation process than did the corresponding E. coli complexes. At one promoter, B. subtilis E sigma A and E. coli E sigma 70 behaved similarly, forming stable open complexes in the absence of any nucleoside triphosphates.

Bacillus subtilis↗