Construction and characterization of E. coli K12 strains in which the transcription of selected genes is desynchronized from translation.
In Escherichia coli, synthesis and translation of individual mRNAs are usually synchronous, so that no long ribosome-free mRNA stretch exists between the RNA polymerase and the leading ribosome. By comparing situations in which the same mRNA (the lacZ mRNA) is synthesized either by the genuine E. coli RNA polymerase or the faster T7 RNA polymerase, we have previously shown that the outpacing of ribosomes by RNA polymerase destabilizes mRNAs, and more so as outpacing becomes larger. This destabilization requires the noncatalytic C-terminal region of RNase E; more generally, there is circumstantial evidence that this region is specifically involved in the fast decay of various untranslated mRNAs. The genetic system designed for desynchronizing transcription and translation with T7 RNA polymerase was originally designed in the E. coli B strain BL21(DE3). Here, we describe procedures for transferring this system to the more common E. coli K12 background. We also show that it can be used as a screen for identifying factors involved in the instability of untranslated mRNA. Protocols in use in this laboratory for RNA extraction, Northern blotting, and beta-galactosidase assay are described and critically discussed.
Proux
Florence
F
Ecole Normale Supérieure, Laboratoire de Génétique Moléculaire, Paris, France.
Dreyfus
Marc
M
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D017931:DNA Primers; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D007763:Lac Operon; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D017931:DNA Primers; D004722:Endoribonucleases; C022537:ribonuclease E