RNA Journal Club 5/13/10
Sergey Proshkin, A. Rachid Rahmouni, Alexander Mironov, Evgeny Nudler
Science, Vol. 328. no. 5977: pp. 504 – 508, 23 April 2010.
This week’s smart summary and analysis by Pavan Vaidyanathan:
In this paper, the authors propose a mechanism for active physical cooperation between an elongating RNA polymerase (RNAP) and a translating ribosome following the polymerase on the transcribed message.
It has been known for several decades that in prokaryotes the processes of transcription and translation are coupled and occur simultaneously. Ribosomes are loaded on to a message as it is getting transcribed by RNAP. This coupled process serves to maximize efficiency of protein synthesis and allows for rapid changes in gene expression. It is also well established that the coupling of translation to transcription can serve as a means of regulation of RNAP. For instance, if the rate of translation is not as high as the rate of transcription, the increasing distance between the ribosome and RNAP allows for the transcription terminator Rho to bind the message and cause RNAP dissociation from the message. Additionally, numerous amino acid biosynthesis operons are regulated by the mechanism of transcription attenuation in which the rate of translation determines the formation of secondary structure behind RNAP that either allows the continuation of transcription or terminates transcription. In this paper however, the authors propose a novel means of regulation of transcription that depends on a physical interaction between RNAP and the first trailing ribosome behind the polymerase.
The authors first showed that in vivo, the ratio of the rate of transcription (nt/sec) to the rate of translation (aa/sec) is consistently ~3.0 in a variety of environmental conditions and stages of growth. Based on this observation, the authors hypothesize that the rate of transcription in vivo is determined by the rate of translation. To test this, they determined the rate of transcription of a plasmid-derived lacZ message in cells grown in media containing low concentrations of ribosome-binding antibiotic (chloramphenicol). They observed that slowing down the ribosome also slowed down the rate of transcription. They confirmed this by testing the rate of transcription of various messages harboring increasing amounts of rare codons, which are expected to slow down translation. As expected, the rate of transcription was inversely proportional to the percentage of rare codons in the message.
RNAP, like other polymerases, is known to backtrack on a message quite frequently. However, the presence of multiple polymerases on the same message significantly decreased backtracking. The authors hypothesized that the ribosome could improve the efficiency of transcription by serving as a physical block to backtracking and ‘forcing’ the polymerase to go forward. In order to test their model, the authors developed an assay to monitor RNAP backtracking in vivo. The assay employed the single-stranded DNA binding probe, chloroacetaldehyde, to monitor the migration of the transcription bubble. They observed that when a single RNAP molecule was forced to encounter a roadblock (lac repressor bound to DNA), it paused and backtracked. However, when there were two RNAP elongation complexes transcribing the same message, this backtracking was reduced substantially. Similarly, the presence of a ribosome behind RNAP also significantly reduced the incidence of backtracking suggesting that the ribosome could improve transcription by preventing backtracking of the leading elongation complex (EC). Using a similar roadblock system, the authors additionally showed by Northern blot analysis of the transcribed mRNA that the leading EC could had much higher rates of readthrough of the roadblock when it was followed by a second EC or by a ribosome.
The authors conclude that the ribosome directly controls the rate of transcription by preventing RNAP backtracking. Because of this cooperation, the rate of transcription is determined by codon usage and nutrient availability as sensed by the ribosome thus allowing precise regulatory adjustment of transcription to translational needs.
Citation for researchblogging.org:
Proshkin S, Rahmouni AR, Mironov A, & Nudler E (2010). Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science, 328 (5977), 504-8 PMID: 20413502