Regulation of protease activity of Escherichia coli RecA protein: A study of novel protease-constitutive recA mutants
Abstract
The RecA protein of Escherichia coli is a relatively small protein but plays an important role in many cellular functions. To understand how this small protein carries out so many functions, a mutational approach was undertaken to dissect its functions and to relate specific functions to specific mutations. The protease-constitutive (Prt$\sp{\rm c}$) recA mutants were especially useful for this purpose, because these function-enhanced mutants could produce proteins specifically altered at functional sites without eliciting a global change of the protein structure. The mutational changes of several of the recA(Prt$\sp{\rm c}$) mutants were determined by DNA sequence analysis and were found to cluster at three regions on the linear RecA polypeptide (N-terminal, middle, and C-terminal). These regions were shown to be involved in both nucleoside triphosphate (NTP) and polynucleotide binding. By DNA sequence analysis of a unique class of recA(Prt$\sp{\rm c}$) mutants, Prt$\sp{\rm c}$Rec$\sp-$ (recombinase negative), I also found that the functional domains for protease and recombinase activities of RecA overlap. To find out why recA(Prt$\sp{\rm c}$) mutants have constitutive RecA protease activities in vivo and why the Prt$\sp{\rm c}$ activities of some mutants are relatively more resistant to inhibition by cytidine plus guanosine (C + G) than that of others, I studied the in vitro biochemical properties of two strong RecA(Prt$\sp{\rm c}$) mutant proteins, RecA1202 and RecA1211. I found that these two mutant proteins, in contrast to RecA$\sp+$ protein, can efficiently use RNA and an enlarged variety of NTPs (CTP, dCTP, GTP, dGTP, UTP, and TTP) as cofactors in the cleavage of LexA repressor. I also found that these mutant proteins show higher affinity for ATP and single-stranded DNA and higher repressor cleavage activities than does RecA$\sp+$ protein. These results provide a more complete explanation than had been available for why recA(Prt$\sp{\rm c}$) mutants have constitutive RecA protease activity. They also explain, for the first time, why C + G can inhibit Prt$\sp{\rm c}$ activities of some recA(Prt$\sp{\rm c}$) mutants are more resistant to C + G inhibition than that of others.
Degree
Ph.D.
Advisors
Tessman, Purdue University.
Subject Area
Molecular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.