Codon-usage based regulation of colicin K synthesis by the stress alarmone ppGpp.
The molecular mechanism of the upregulation of Escherichia coli colicin K (Cka) synthesis during stress conditions was studied. Nutrient starvation experiments and the use of relA spoT mutant strains, IPTG-regulated overproduction of ppGpp and lacZ fusions revealed that the stringent response alarmone guanosine 3',5'-bispyrophosphate (ppGpp) is the main positive effector of Cka synthesis. Comparison of the amounts of protein produced (Western blotting) and specific mRNA (Northern blotting) before and after nutrient starvation demonstrated increases in Cka protein with unaltered specific mRNA levels, suggesting a post-transcriptional regulatory mechanism. Reporter (beta-galactosidase) assays using truncated cka of variable length fused to lacZ located the key regulatory region close to the 5' end of the cka mRNA. Closer analysis of this region indicated the presence of several rare codons, including the leucine-encoding codon CUA. Synonymous exchange of the rare codons with more frequently used ones abolished the regulatory effect of ppGpp. Supplementation of the strain with the plasmid CodonPlus carrying several rare tRNA genes yielded similar results, indicating that codon usage (in particular, the fifth codon for the amino acid leucine) and tRNA availability (i.e. tRNAleu) are the key elements of the regulatory function of ppGpp. We conclude that ppGpp regulates Cka synthesis via a novel post-transcriptional mechanism that is based on rare codon usage and variable cognate tRNA availability.
Kuhar
I
I
Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia.
van Putten
J P
JP
Zgur-Bertok
D
D
Gaastra
W
W
Jordi
B J
BJ
D003062:Codon / Q000235:genetics*; D003087:Colicins / Q000096:biosynthesis* / Q000235:genetics; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006159:Guanosine Tetraphosphate / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D014158:Transcription, Genetic
D003062:Codon; D003087:Colicins; D003470:Culture Media; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D006159:Guanosine Tetraphosphate