Ribosome reactivates transcription by physically pushing RNA polymerase out of transcription arrest

Abstract

In bacteria, the first two steps of gene expression-transcription and translation-are spatially and temporally coupled. Uncoupling may lead to the arrest of transcription through RNA polymerase backtracking, which interferes with replication forks, leading to DNA double-stranded breaks and genomic instability. How transcription-translation coupling mitigates these conflicts is unknown. Here we show that, unlike replication, translation is not inhibited by arrested transcription elongation complexes. Instead, the translating ribosome actively pushes RNA polymerase out of the backtracked state, thereby reactivating transcription. We show that the distance between the two machineries upon their contact on mRNA is smaller than previously thought, suggesting intimate interactions between them. However, this does not lead to the formation of a stable functional complex between the enzymes, as was once proposed. Our results reveal an active, energy-driven mechanism that reactivates backtracked elongation complexes and thus helps suppress their interference with replication.

Keywords: RNA polymerase; backtracking; conflicts with replication; ribosome; transcription–translation coupling.

Fig. 1.

Ribosomes push backtracked RNAP. (A) Initial steps of assembly of the coupled transcription–translation system. Purified translation initiation complexes (M complexes) are coupled to an assembled transcription EC and immobilized on streptavidin beads, and the EC is allowed to transcribe to the position that causes stable backtracking. (B and C) Scheme and results of the experiment. EC coupled to translation M complex is walked to a position that causes stable backtracking (because of collision with a streptavidin bead). Then ribosome translates an 11-aa peptide approaching the backtracked EC before it stalls with a UAG stop codon in the A-site (lanes 5 to 8). The positions of RNAP and the ribosome are determined by GreB and RelE cleavage on 5′ radiolabeled mRNA, respectively.

Fig. 2.

The distance between the coupled ribosome and RNAP interacting on mRNA. (A and B) Scheme and results of the experiment. MF complexes are coupled to ECs as in Fig. 1A, except here the EC is walked to the abasic position, where it is stabilized in the posttranslocated state (SI Appendix, Fig. S3A) and cannot extend mRNA further. mRNAs contain UAG stop codons (to standardize efficiency by RelE cleavage among mRNAs) at different distances from the RNAP active center. Ribosomes translate 9-, 10-, 11-, or 12-aa peptides toward the EC before they reach UAG stop codons. The availability of UAG in the A-site, as a measure of successful translocation of the ribosome, is tested by RelE cleavage (lanes 4, 8, 12, and 16). Ribosomes that came in contact with RNAP and cannot translocate have peptidyl-tRNA in the A-site, making them inaccessible for RelE (lane 16). (C) Efficiencies of RelE cleavage at UAGs located at various distances from the RNAP active center. The positions of one, two, and three nucleotide deletions and insertions in mRNAs are shown in A and in SI Appendix, Fig. S1A. Data points are means and error bars are SDs from 3 to 13 experiments. Black squares are the RelE cleavage efficiencies on the corresponding mRNAs when the EC was walked 8 bp (on a template without abasic site) further away from stop codons. (D) The distances on mRNA between the RNAP active center and the ribosome’s PTC in both the “strained” contact, when the ribosome is forced into a stalled EC (Bottom), and the “nonstrained” contact, when ribosome translocation pushes the backtracked RNAP (Top) are the same, 25 to 26 nucleotides.

Fig. 3.

Models of transcription/translation coupling. The cooperation model implies contact between the ribosome and RNAP during pushing but no formation of a stable functional complex. However, cooperation may take place with or without formation of a structural supercomplex between the ribosome and EC.