Control of the tRNA-tufB operon in Escherichia coli. 2. Mechanisms of the feedback inhibition of tufB expression studied in vivo and in vitro.
The mechanism underlying feedback inhibition of tufB expression has been studied in vivo by gene-dosage experiments and by gene and operon fusions involving lacZ. Raising the cellular EF-Tu content, by introducing a multicopy plasmid encoding EF-TuA into the cell, repressed the level of EF-TuB but left the content of tRNA(Thr)3, encoded by the tRNA-tufB operon, unaffected. This indicates that autoregulation of chromosomal tufB expression does not occur by modulating transcription initiation at the promoter of the tRNA-tufB operon. This conclusion is further substantiated by experiments with a tRNA':lacZ operon fusion. The molecular ratio of chromosome-borne tufA and tufB transcripts also remained unaltered under conditions of excess EF-Tu, though experiments with a tRNA-tufB':lacZ operon fusion showed a decrease of tufB transcripts. Our data further exclude drastic effects of the autogenous repressor on processing of the contranscript of the operon into monocistronic tufB RNA and on alteration of EF-TuB turnover. Two possible mechanisms remain, which cannot yet be decided between. One is modulation of EF-Tu by transcription termination either directly or indirectly by affecting antitermination. The second is translational repression. In vitro translation of transcripts derived from SP6 clones did not reveal any feedback inhibition of EF-TuB synthesis. Surprisingly, addition of EF-Tu to a coupled transcription/translation systems was found to block transcription initiation at the primary promoter of the tRNA-tufB operon by over 90%. Although this in vitro effect of EF-Tu could not be demonstrated in vivo, possibly because of a difference in higher-order structure between plasmid-borne and chromosome-borne DNA, it indicates that under certain conditions EF-Tu binds very specifically to the tRNA-tufB operon promoter or its upstream region.
Van Delft
J H
JH
Department of Biochemistry, University of Leiden, The Netherlands.
Talens
A
A
De Jong
P J
PJ
Schmidt
D S
DS
Bosch
L
L
D003001:Cloning, Molecular; D004278:DNA, Superhelical / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005246:Feedback; D005786:Gene Expression Regulation; D005796:Genes; D005798:Genes, Bacterial; D009876:Operon; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004278:DNA, Superhelical; D012329:RNA, Bacterial; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu