Author/Editor		Kuhar, Irena; van Putten, Jos PM; Žgur-Bertok, Darja; Gaastra, Wim; Jordi, Bart JAM
Title		Codon-usage based regulation of colicin K synthesis by the stress alarmone ppGpp
Type		članek
Source		Mol Microbiol
Vol. and No.		Letnik 41, št. 1
Publication year		2001
Volume		str. 207-16
Language		eng
Abstract		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 IacZ fusions revealed that the stringent response alarmone guanosine 3',5'-bispyrophosphate (ppGpp) is the main positive effector of Cka synthesis. Comparison of the arnoun4s o9 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 off several rare codons, including the leucine encoding codon CUA. Synonymous exchange of Ehe rare colons with more frequently used ones abolished the regulatory effect of ppGpp. SupplemenPation of the strain with the plasmid CodonPlus carrying several rare tRNA genes yielded similar results, indicating that colon usage (in particular, the fifth colon for the amino acid leucine) and tRNA availablity (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 colon usage and variable cognate tRNA availability.
Descriptors		ESCHERICHIA COLI COLICINS STARVATION CODON GUANOSINE DIPHOSPHATE RNA, MESSENGER BACTERIAL PROTEINS BETA-GALACTOSIDASE PLASMIDS RNA, TRANSFER, LEU