Peer-Reviewed Journal Details
Mandatory Fields
Butler, D,Fitzgerald, GF;
2001
January
Journal of Bacteriology
Transcriptional analysis and regulation of expression of the ScrFI restriction-modification system of Lactococcus lactis subsp cremoris UC503
Validated
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Optional Fields
PROMOTER REGION ECORII METHYLTRANSFERASE DNA-METHYLTRANSFERASE LLAI RESTRICTION GENE-EXPRESSION BINDING PROTEIN ENDONUCLEASE SEQUENCES CLONING
183
4668
4673
ScrFI is a type II restriction-modification system from Lactococcus lactis which recognizes the nucleotide sequence 5 ' -CC down arrow NGG-3 ', cleaving at the point indicated by the arrow, and it comprises an endonuclease gene that is flanked on either side by genes encoding two 5-methylcytosine methylases. An open reading frame (orfX) of unknown function is located immediately upstream of these genes, In this study Northern analysis was performed, and it revealed that orfX, scrFIBM, and scrFIR are cotranscribed as a single polygenic mRNA molecule, while scrFIAM is transcribed independently, 5 ' extension analysis indicated that the start site for the scrFIAM promoter was a thymine located 4 bp downstream of the -10 motif, The transcriptional start site for the orfX promoter was also found to be a thymine which is more atypically located 24 bp downstream of the -10 motif proximal to the start codon, A helix-turn-helix motif was identified at the N-terminal end of one of the methylases (M,ScrFIA), In order to determine if this motif played a role in regulation of the ScrFI locus, M,ScrFIA was purified. It was then employed in gel retardation assays using fragments containing the two promoters found on the ScrFI operon, one located upstream of orfX and the other located just upstream of scrFIAM. M.ScrFIA was found to hind to the promoter region upstream of the gene encoding it, indicating that it may have a regulatory role. In further studies the two putative promoters were introduced into a vector (pAK80) upstream of a promoterless lacZ gene, and cloned fragments of the ScrFI locus were introduced in trans with each of these promoter constructs to investigate the effect on promoter activity. These results implicated M,ScrFIA in regulation of both promoters on the ScrFI locus.
Grant Details