Effect of the pcnB mutation on functional mRNA stability and protein synthesis. (A) Cultures of N3433 and IBPC 903 (pcnB) were grown in MOPS medium containing glucose and amino acids lacking methionine and cysteine until mid-log phase. Cells were treated with rifampicin (zero time), and their protein-synthesizing capacity was monitored after 1, 3, 5, 7 and 9 min by pulse-labelling for 1 min with radioactive methionine. Soluble proteins were analyzed on SDS-PAGE. Left panel: Coomassie blue staining; right panel: autoradiograph. The positions of polypeptides overproduced in the pcnB strain are indicated by an arrow for the 66-kDa protein and by asterisks. (B) Comparison of the protein profile of N3433 (lane 1), CA244 (lane 3) and IBPC 694 (lane 5) wild-type strains to their pcnB::kan^R (IBPC 903 and CA244-PAP, respectively) (lanes 2 and 4) and pcnB80 (IBPC 690) (lane 6) derivatives. Cells were grown in LB medium until mid-log phase and soluble proteins analyzed by SDS-PAGE electrophoresis and revealed by Coomassie Blue staining.

The pcnB mutation increases the amount of GlmS. (A) Protein profile of N3433 (wt) and IBPC 903 (pcnB) strains grown in LB medium were analyzed by 2D-PAGE. Representative gels from experiments performed in triplicate were presented. Soluble proteins with isoelectric point (pI) values in the range 3.0–11.0 and molecular mass in the range of 26–170 kDa were resolved in 12% SDS-PAGE gels and colloidal blue stained for comparative analysis. Corresponding sections of representative gels show the increased synthesis of two spots in the pcnB strain compared with the wt. The two spots identified as GlmS by MALDI-TOF are indicated by arrows. Protein level were determined by densitometric quantification (PDQuest™ 2D analysis software, Biorad). (B) Western blot showing the GlmS polypeptide synthesized in wt and its pcnB derivative. Total proteins were loaded on 10% SDS-PAGE. Position of the GlmS protein used as a control is indicated on the left.

The pcnB mutation increases amounts of the glmS mRNA. Total RNA (12 μg) from N4333 (wt) and IBPC 903 (pcnB) strains grown in LB medium at 37°C were analyzed on Northern blot and probed for the glmS mRNA (upper part). Reprobing of the same membrane with the 5S rRNA probe is shown on the lower part. Positions of rRNA markers are indicated on the left.

Fate of the transcripts of the glmU-glmS cotranscript. (A) Structure of the glmU-glmS transcript and of the processed mRNAs. The map of the glmU-glmS genes of E.coli shows the location of the 2 glmU promoters (arrows), of the transcription terminator (t) and the major RNase E processing site (black triangle). The structure of the primary transcript is indicated beneath the map, together with the mRNA species that were detected by Northern blot. The Northern blots do not distinguish between mRNAs starting at the two promoters. The small black box locates the oligonucleotide used in the primer extension experiment. Locations of the RNA used to probe for glmU and glmS are indicated by rectangles. (B) Northern blot analysis of transcripts of the glmU-glmS operon. Strains indicated at the top of the autoradiograph were grown at 30°C in LB medium and shifted to 44°C for the times indicated in minutes at the top of the autoradiograph. Total RNA (12 μg) were analyzed on Northern blot, which were probed for glmS, glmU mRNAs and 5S rRNA. 9S rRNA precursors accumulate after RNase E inactivation. As reported previously, rne⁺ strains contain higher amounts of 5S rRNA after the shift to 44°C (51). (C) Overexposure of a Northern blot probed for glmU transcripts. (D) Primer extension analysis of the glmU-glmS intercistronic region. Total RNA (12 μg) from N3433 (wt) and IBPC 903 (pcnB) strains were used as templates for AMV reverse transcriptase with the 5′ labelled glmS oligonucleotide (1 pmol). The extension products were analyzed on a 6% denaturating polyacrylamide gel. Molecular sizing markers are shown on the right side of the autoradiograph. The glmS primer was used with the Sequenase kit (Amersham) on a PCR amplified fragment to precisely locate the 5′ extremities of the reverse transcriptase reactions (data not shown).

PAP I destabilizes the glmS transcript. (A) Autoradiograph comparing the decay kinetics of glmS mRNA in strains N3433 (wt) and IBPC 903 (pcnB). Times after inhibition of transcription at which glmS (upper part) and 5S (lower part) RNA contents were determined are indicated above each lane. (B) RNA levels were quantified by phosphorimagery and plotted as a function of time. Quantification of the results.