Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes. Expression from reporter genes does not always reflect functional mRNA levels.
Induction of beta-galactosidase from high copy-number plasmids was found to reduce the synthesis of other cellular proteins in Escherichia coli. The reduction depends on the protein in question and on the induction level of the beta-galactosidase. It could be observed transiently within one minute after induction and in some cases also during steady-state induction. Our interpretation is that the concentration of the free ribosomal subunits decreases after induction, leading to an increased competition among the individual ribosome binding sites for ribosomes. The immediate reduction in the synthesis individual proteins after induction of beta-galactosidase was used as an assay to measure in vivo the efficiency of a ribosome binding site. These efficiencies were compared to the calculated affinities between the ribosome binding site of specific mRNA species and the 3' end of 16 S RNA. For several mRNAs with similar Shine-Dalgarno sequences, the sensitivity to competition differed twofold. Our results show, that both transiently during induction of lacZ and also at very high steady-state expression levels, the expression from reporter genes, including the lacZ gene itself, does not reflect the levels of the mRNAs in a simple way.
Vind
J
J
Institute of Microbiology, University of Copenhagen, Denmark.
Sørensen
M A
MA
Rasmussen
M D
MD
Pedersen
S
S
D001426:Bacterial Proteins / Q000096:biosynthesis*; D001483:Base Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D007763:Lac Operon; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger