Mutagenesis and functional analysis of the Escherichia coli tRNA(1Leu) promoter.
The leuV promoter which produces tRNA(1Leu) in Escherichia coli has been extensively mutagenized in order to determine the effects of altered sequences on promoter efficiency (strength) and on growth-rate-dependent regulation (GDR). Each mutant promoter was ligated with a beta-galactosidase reporter gene into the chromosome of a host cell by phage lambda lysogenization. Reporter gene activities were measured for cells growing in selected media at various growth rates. Sequences which flank the -10 consensus region, when altered, caused remarkable up-promoter effects, increasing efficiency in some cases almost 10-fold. One up mutation which had five successive T residues in the 'discriminator' region completely abolished GDR, whereas several mutations with single base changes in the discriminator had little or no effect on GDR. Another mutation which changed one base in the -35 region to bring it to consensus increased promoter strength 18-fold and sharply reduced GDR. Chimaeric promoters in which segments of leuV were replaced by segments of the his operon showed that only when the discriminator of leuV is replaced by the his discriminator was GDR-disturbed. All upstream sequences which were replaced by his sequences had little effect on GDR. Overall, there appeared to be little correlation between promoter efficiency and GDR.
Bauer
B F
BF
Department of Microbiology, Virginia Commonwealth University and Medical College of Virginia, Richmond 23298-0678.
Elford
R M
RM
Holmes
W M
WM
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics*; D012356:RNA, Transfer, Leu / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion
D012329:RNA, Bacterial; D012356:RNA, Transfer, Leu; D011993:Recombinant Fusion Proteins