Biomedical subjects

Gerald B Koudelka

Publications and source records attributed to Gerald B Koudelka.

5 recordsLinked to original sources

PubMed2004

Monovalent cations regulate DNA sequence recognition by 434 repressor.

The bacteriophage 434 repressor distinguishes between its six naturally occurring binding sites using indirect readout. In indirect readout, sequence-dependent differences in the structure and flexibility of non-contacted bases in a protein's DNA-binding site modulate the affinity of DNA for protein. The conformation and flexibility of a DNA sequence can be influenced by the interaction of the DNA bases or backbone with solution components. We examined the effect of changing the cation-type present in solution on the stability and structure of 434 repressor complexes with wild-type and mutant OR1 and OR3, binding sites that differ in their contacted and non-contacted base sequences. We find that the affinity of repressor for OR1, but not for OR3, depends remarkably on the type and concentration of monovalent cation. Moreover, the formation of a stable, specific repressor-OR1 complex requires the presence of monovalent cations; however, repressor-OR3 complex formation has no such requirement. Changing monovalent cation type alters the ability of repressor to protect OR1, but not OR3, from *OH radical cleavage. Altering the relative length of the poly(dA) x poly(dT) tract in the non-contacted regions of the OR1 and OR3 can reverse the cation sensitivity of repressor's affinities for these two sites. Taken together these findings show that cation-dependent alterations in DNA structure underlies indirect readout of DNA sequence by 434 repressor and perhaps other proteins.

Base Sequence↗

PubMed2004

The preferred substrate for RecA-mediated cleavage of bacteriophage 434 repressor is the DNA-bound dimer.

Induction of a lysogen of a lambdoid bacteriophage usually involves RecA-stimulated autoproteolysis of the bacteriophage repressor protein. Previous work on the phage repressors showed that the monomeric form of the protein is the target of RecA. Our previous work indicated that in the case of bacteriophage 434, virtually none of the repressor is present as a monomer in vivo. Hence, if the repressor in a lysogen is present as a dimer, how can RecA-stimulated autoproteolysis play a role in bacteriophage induction? We examined this question by determining the rate of RecA-stimulated 434 repressor cleavage as a function of repressor concentration and added DNA. Our results show that binding of 434 repressor to a specific DNA binding site dramatically increases the velocity of repressor autocleavage compared to the velocity of cleavage of the monomer and concentration-induced dimer. DNA binding-deficient hemidimers formed between the intact repressor and its C-terminal domain fragment have a lower rate of cleavage than DNA-bound dimers. These results show that the DNA-bound 434 repressor dimer, which is the form of the repressor that is required for its transcriptional regulatory functions, is the preferred form for RecA-stimulated autocleavage. We also show that the rate of repressor autocleavage is influenced by the sequence of the bound DNA. Kinetic analysis of the autocleavage reaction indicated that the DNA sequence influences the velocity of 434 repressor autocleavage by affecting the affinity of the repressor-DNA complex for RecA, not the chemical cleavage step. Regardless of the mechanism, the finding that the presence and precise sequence of DNA modulate the autocleavage reaction shows that DNA allosterically affects the function of 434 repressor.

Bacteriophage lambda↗

PubMed2004

Purification and characterization of the repressor of the shiga toxin-encoding bacteriophage 933W: DNA binding, gene regulation, and autocleavage.

The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding Escherichia coli (STEC) strains, are carried on lambdoid prophages resident in all known STEC strains. The stx genes are expressed only during lytic growth of these temperate bacteriophages. We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein. Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (OR). Also typical, when bound at OR, 933W repressor functions as an activator at the PRM promoter and a repressor at the PR promoter. In contrast to other lambdoid bacteriophages, 933W left operator (OL) contains only two repressor binding sites, but the OL-bound repressor still efficiently represses PL transcription. Lambdoid prophage induction requires inactivation of the repressor's DNA binding activity. In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a "linker " region that joins the two domains of these proteins. However, 933W repressor protein contains neither an Ala-Gly nor an alternative Cys-Gly dipeptide cleavage site anywhere in its linker sequence. We show here that the autocleavage occurs at a Leu-Gly dipeptide. Thus, the specificity of the repressor autocleavage site is more variable than thought previously.

Bacteriophage lambda↗

PubMed2003

DNA-stimulated assembly of oligomeric bacteriophage 434 repressor: evidence for cooperative binding by recruitment.

Typical of many transcriptional regulatory proteins, the lambdoid bacteriophage repressors bind cooperatively to multiple sites on DNA. This cooperative binding is essential for establishment and maintenance of phage lysogeny. In the phage, two repressor homodimers, one bound at each of the adjacent operator sites, interact to form the tetramer that is necessary for the cooperative binding of the repressor. Bacteriophage 434 repressor does not form tetramers in the absence of DNA, and the mechanism by which the tetramer assembles on the two adjacent sites is unknown. Hence DNA binding may stimulate the repressor to form tetramers and formation of a repressor oligomer (> or = 3 monomers) on a single DNA sites may precede multisite binding. Consistent with these ideas, a complex containing three repressor molecules readily assembles on a single operator (O(R)1) site. Mutations that inhibit cooperative tetramer binding to the adjacent O(R)1 and O(R)2 sites also block formation of this complex. Together with other evidence, these findings show that the complex that forms on a single site assembles using the same interface as does the tetramer assembled on adjacent operator sites. Adding additional O(R)1 DNA dissociates the oligomeric repressor-DNA complexes into dimeric repressor-O(R)1 complexes. In contrast, adding O(R)2 to these complexes results in the formation of a repressor oligomer containing an O(R)2 and an O(R)1 site. The observation that a repressor oligomer bound to two O(R)1 sites is less stable than the one formed between repressor dimers bound to O(R)1 and O(R)2 implies that DNA allosterically influences the structure of the 434 repressor. Together these findings suggest that an O(R)1-bound repressor may cooperatively help repressor bind to O(R)2 by recruiting an additional repressor molecule from solution that subsequently occupies O(R)2.

Bacteriophages↗

PubMed2003

The role of the minor groove substituents in indirect readout of DNA sequence by 434 repressor.

The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89-91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH(2) group on the purine bases at the binding site center. The N2-NH(2) group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH(2) group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface.

Amino Acid Sequence↗