In vivo dynamics of intracistronic transcriptional polarity.
Transcriptional polarity occurs in Escherichia coli when cryptic Rho-dependent transcription terminators become activated as a consequence of reduced translation. Increased spacing between RNA polymerase and the leading ribosome allows the transcription termination factor Rho to bind to mRNA, migrate to the RNA polymerase, and induce termination. Transcriptional polarity results in decreased synthesis of inefficiently translated mRNAs and, therefore, in decreased expression not only of downstream genes in the same operon (intercistronic polarity) but also of the cistron in which termination occurs (intracistronic polarity). To quantitatively measure the effect of different levels of translation on intracistronic transcription termination, the polarity-prone lacZ reporter gene was fused to a range of mutated ribosome binding sites, repressed to different degrees by local RNA structure. The results show that polarity gradually increases with decreasing frequency of translational initiation, as expected. Closer analysis, with the help of a newly developed kinetic model, reveals that efficient intracistronic termination requires very low translational initiation frequencies. This finding is unexpected because Rho is a relatively small protein that binds rapidly to its RNA target, but it appears to be true also for other examples of transcriptional polarity reported in the literature. The conclusion must be that polarity is more complex than just an increased exposure of the Rho binding site as the spacing between the polymerase and the leading ribosome becomes larger. Biological consequences and possible mechanisms are discussed.
de Smit
Maarten H
MH
Section Genexpress, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, PO Box 9502, 2300 RA Leiden, The Netherlands. m.smit@chem.leidenuniv.nl
Verlaan
Paul W G
PW
van Duin
Jan
J
Pleij
Cornelis W A
CW
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D012333:RNA, Messenger