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.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Transcription elongation stalls at lesions in the DNA template
. For the DNA lesion to be repaired, the stalled transcription elongation complex (EC) has to be removed from the damaged site
. Here we ...show that translation, which is coupled to transcription in bacteria, actively dislodges stalled ECs from the damaged DNA template. By contrast, paused, but otherwise elongation-competent, ECs are not dislodged by the ribosome. Instead, they are helped back into processive elongation. We also show that the ribosome slows down when approaching paused, but not stalled, ECs. Our results indicate that coupled ribosomes functionally and kinetically discriminate between paused ECs and stalled ECs, ensuring the selective destruction of only the latter. This functional discrimination is controlled by the RNA polymerase's catalytic domain, the Trigger Loop. We show that the transcription-coupled DNA repair helicase UvrD, proposed to cause backtracking of stalled ECs
, does not interfere with ribosome-mediated dislodging. By contrast, the transcription-coupled DNA repair translocase Mfd
acts synergistically with translation, and dislodges stalled ECs that were not destroyed by the ribosome. We also show that a coupled ribosome efficiently destroys misincorporated ECs that can cause conflicts with replication
. We propose that coupling to translation is an ancient and one of the main mechanisms of clearing non-functional ECs from the genome.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Abstract
Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation ...by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3′CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1’s accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.
Inhibitory compounds of peptidoglycan deacetylase PgdA identified by virtual HTS and confirmed by novel enzyme assay.
The essential cell wall peptidoglycan is the target of several components of the ...innate immune system and its disruption results in lysis of invading bacteria. The pathogen
Streptococcus pneumoniae produces a peptidoglycan N-acetylglucosamine deacetylase, PgdA, to modify the peptidoglycan structure. The activity of PgdA contributes to the bacteria's resistance to lysozyme, which is an important antimicrobial factor of the human innate immune system. In this study we report on the activity of PgdA against natural and artificial substrates. We have also established a virtual high-throughput screening and a new enzyme assay to search for compounds inhibiting PgdA. Two compounds with IC
50 values in the micromolar range have been identified and they could serve as leads for the search of inhibitors of PgdA, an important pneumococcal virulence factor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Transcription and translation form the basis of gene expression in all cells. In prokaryotes they are linked both spatially and temporally as the ribosomes begin translation of the RNA before the ...RNAP has finished transcribing the entire region, a process known as coupling. Interplay between the two machineries is highly complex and plays an important role in gene expression. To date, most of the studies into transcription-translation coupling have been carried out in vivo, and have focused on the indirect interactions such as attenuation. Due to the many accessory factors for both transcription and translation present within the cell, there is currently no known technique to study direct interactions between the RNAP and the ribosome. Recently, an in vitro transcription-translation system was developed in our lab that is formed from only the pure components required for transcription and translation. This allows the stepwise control of the RNAP and the ribosome. The aim of this study was to determine how close the RNAP and the ribosome can become on the same nascent RNA. The coupled in vitro system was redesigned and optimised to measure the distance between the actively transcribing RNAP and the ribosome translating the same transcript. We show that the ribosome can approach the RNAP as close as 26 nts between the A-site of the ribosome and the active site of the RNAP. This distance is far shorter than was previously thought and reveals a very close contact between the two machineries.
Transcription and translation form the basis of gene expression in all cells. In prokaryotes they are linked both spatially and temporally as the ribosomes begin translation of the RNA before the ...RNAP has finished transcribing the entire region, a process known as coupling. Interplay between the two machineries is highly complex and plays an important role in gene expression. To date, most of the studies into transcription-translation coupling have been carried out in vivo, and have focused on the indirect interactions such as attenuation. Due to the many accessory factors for both transcription and translation present within the cell, there is currently no known technique to study direct interactions between the RNAP and the ribosome. Recently, an in vitro transcription-translation system was developed in our lab that is formed from only the pure components required for transcription and translation. This allows the stepwise control of the RNAP and the ribosome. The aim of this study was to determine how close the RNAP and the ribosome can become on the same nascent RNA. The coupled in vitro system was redesigned and optimised to measure the distance between the actively transcribing RNAP and the ribosome translating the same transcript. We show that the ribosome can approach the RNAP as close as 26 nts between the A-site of the ribosome and the active site of the RNAP. This distance is far shorter than was previously thought and reveals a very close contact between the two machineries.
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