| Abstract [eng] |
The regB gene of T4 – related bacteriophages codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG or, in a few cases, GGAU tetranucleotide making it a unique site-specific restriction endoribonuclease. Endoribonuclease RegB of phage T4 have been studied for almost 20 years, while the homologues of RegB from phylogeneticaly distant T4-type phages deserved only initial studies. In this study, four temperature sensitive mutants of bacteriophages RB49 and RB69 endoribonuclease RegB have been constructed. The activities of RegB mutants RB69RegB_M11, RB69RegB_M41 and RB49RegB_ts2, as determined by primer extension analysis of regB transcripts, were detectable at 30ºC and decreased markedly at 42ºC temperature. We found that these mutants were less toxic to Escherichia coli cells at 42ºC than at 30ºC. The regB_ts2 gene, coding for the most clear RegB temperature sensitive mutant, was cloned into the vector pLT532, which carries lacZ gene fused with the Shine-Dalgarno region of T4 gene 30.7 carrying RegB cleavage site. We found that RegB_ts2 cleaves the lacZ transcripts induced from this plasmid and regulates its translation. |
