Disruption of transcription–translation coordination in Escherichia coli leads to premature transcriptional termination

Abstract

Introductory paragraph Tight coordination between transcription and translation is crucial to maintaining the integrity of gene expression in bacteria. How bacteria manage to coordinate these two processes remains unclear. Possible direct physical coupling between the RNA polymerase and ribosome has been intensely investigated in recent years. Here, we quantitatively characterize the transcriptional kinetics of E. coli in different growth conditions. Transcriptional and translational elongation remain coordinated under various nutrient conditions as previously reported. However, transcriptional elongation was not affected under antibiotics that slowed down translational elongation. The same was found by introducing nonsense mutation that completely dissociated transcription from translation. Our data thus provide direct evidences showing that translation is not required to maintain the speed of transcriptional elongation. In cases where transcription and translation are dissociated, our study provides quantitative characterization of the resulting process of premature transcriptional termination (PTT). PTT-mediated polarity caused by translationtargeting antibiotics substantially affected the coordinated expression of genes in several long operons studied, contributing to important physiological effects of these antibiotics. Our results also suggest a model in which the coordination between transcriptional and translational elongation under normal growth conditions is implemented by ppGpp.

A Decrease in Transcription Capacity Limits Growth Rate upon Translation Inhibition

“…In summary, regulation of transcription initiation by ppGpp cannot solely explain the change in GFP expression from different promoters with increasing sublethal levels of chloramphenicol. However, the shared increase in GFP concentration observed for both the P1 and P5 promoters is consistent with an overall increase in the translation rate, as observed previously in these experimental conditions (19,21).…”

“…By the same mechanism, increased probability of ribosomes stalling in the presence of chloramphenicol can also lead to degradation of a whole operon's RNA, so that genes that are found farther downstream from the transcription start site have a higher probability of not being translated than those at the beginning of the operon. A recent study has shown this to be the case for the genes within the lac operon and those in the ribosomal protein operons S10 and Spc (21). The rpoB and rpoC genes follow each other within the same operon, which can result in a further decrease in the expression of the downstream gene.…”

“…Increased expression from the P1 promoter in the growth media lacking cAA would be expected from a decrease in ppGpp as a result of increased amino acid pools due to ribosome inhibition; the P5 promoter on the other hand does not contain the GC-rich discriminator region and is not expected to show increased activity upon a decrease in ppGpp. ppGpp, however, is also known to negatively regulate the activity of ribosomes and the overall translation rate as well as the transcription elongation rate (7,8,21); therefore, a decrease in ppGpp in the growth media lacking cAA is expected to lead to an increase in translation rate, irrespective of the promoter sequence. The similar increase in GFP concentration of these two different promoters thus points to a stronger effect of ppGpp on GFP expression at the level of translation and transcription elongation of the gfpmut2 gene rather than at the transcription initiation level.…”

“…This has been proposed to result from a decrease in the resources available for the production of nonribosomal proteins that become limiting for cellular metabolism (18). More recent results point to a decrease in the fraction of active ribosomes to explain the decrease in the total protein production rate (19,21).…”

Structural basis of transcription-translation coupling and collision in bacteria

“…We conclude that coupling of NusG to the ribosome requires the P-site to be more than 34 nucleotides from the 3' end of the mRNA. 6 The arrangement of RNAP and ribosome in our structure resembles the expressome formed by collision of translating ribosomes with stalled RNAP (RNAP backbone RMSD ~3Å based on 16S rRNA superposition) (9) ( Fig S8B and S9A,B). We therefore term this molecular state the 'collided expressome'.…”