In vitro experimental system for analysis of transcription–translation coupling

Castro-Roa, Daniel; Zenkin, Nikolay

doi:10.1093/nar/gkr1262

Cited by 33 publications

(44 citation statements)

References 38 publications

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“…The RNA segment that opens between the uncoupled ribosome and EC in vivo has a much lower potential to form stable RNA structures which inhibit backtracking than the unconstrained RNA transcripts that form in vitro . It will be of significant interest to study the mechanistic basis for EC control by DksA using in vitro coupled transcription/translation systems (Castro-Roa and Zenkin, 2012). …”

Section: Discussionmentioning

confidence: 99%

DksA Guards Elongating RNA Polymerase against Ribosome-Stalling-Induced Arrest

et al. 2014

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Summary In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.

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Section: Discussionmentioning

confidence: 99%

DksA Guards Elongating RNA Polymerase against Ribosome-Stalling-Induced Arrest

et al. 2014

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“…To test for translation repression, Meades and coworkers (12) used a commercial E. coli in vitro transcription-translation kit (now discontinued by the supplier). Unfortunately, the resulting data were not quantitated, and as recently pointed out (29), these transcription-translation preparations are designed for protein production and not properly constituted such that all RNA polymerase molecules are coupled to translation and all ribosomes are coupled to nascent mRNAs. Hence, these systems do not accurately mimic the in vivo situation.…”

Section: Discussionmentioning

confidence: 99%

Evidence against Translational Repression by the Carboxyltransferase Component of Escherichia coli Acetyl Coenzyme A Carboxylase

Smith

Cronan

2014

J Bacteriol

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In Escherichia coli, synthesis of the malonyl coenzyme A (malonyl-CoA) required for membrane lipid synthesis is catalyzed by acetyl-CoA carboxylase, a large complex composed of four subunits. The subunit proteins are needed in a defined stoichiometry, and it remains unclear how such production is achieved since the proteins are encoded at three different loci. Meades and coworkers (G. Meades, Jr., B. K. Benson, A. Grove, and G. L. Waldrop, Nucleic Acids Res. 38:1217-1227, 2010, doi:http://dx.doi.org/ 10.1093/nar/gkp1079) reported that coordinated production of the AccA and AccD subunits is due to a translational repression mechanism exerted by the proteins themselves. The AccA and AccD subunits form the carboxyltransferase (CT) heterotetramer that catalyzes the second partial reaction of acetyl-CoA carboxylase. Meades et al. reported that CT tetramers bind the central portions of the accA and accD mRNAs and block their translation in vitro. However, long mRNA molecules (500 to 600 bases) were required for CT binding, but such long mRNA molecules devoid of ribosomes seemed unlikely to exist in vivo. This, plus problematical aspects of the data reported by Meades and coworkers, led us to perform in vivo experiments to test CT tetramermediated translational repression of the accA and accD mRNAs. We report that increased levels of CT tetramer have no detectable effect on translation of the CT subunit mRNAs.

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“…The two systems differ on how they are set-up and are designed to monitor either of the two molecular machineries. The first system, ''transcription-first'' coupled transcription translation system (TR-CTT) was designed to analyze the outcome on translation by observing the synthesized peptide directly whereas the second system, ''translation-first'' coupled transcription translation system (TL-CTT) follows the outcome on transcription by monitoring the RNA transcript [9]. The methodologies for the setting up of these systems will be discussed throughout this article.…”

Section: Introductionmentioning

confidence: 99%

“…Colored dots on gel are under the spots corresponding to the peptide. (À)and (+) represent the cathode and anode respectively[9].…”

mentioning

confidence: 99%

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Methodology for the analysis of transcription and translation in transcription-coupled-to-translation systems in vitro

Castro-Roa

Zenkin

2015

Methods

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The various properties of RNA polymerase (RNAP) complexes with nucleic acids during different stages of transcription involve various types of regulation and different cross-talk with other cellular entities and with fellow RNAP molecules. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of outcomes of gene expression, whereas the study of the physical interaction of the ribosome and the RNAP remains obscure partly due to the lack of a system that allows such observations. In this article we will describe the methodology needed to set up a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing investigation of the interactions between the two machineries at colliding and non-colliding distances. In the same time RNAP can be put in various types of states, such as paused, roadblocked, backtracked, etc. The experimental system thus allows studying the effects of the ribosome on different aspects of transcription elongation and the effects by RNAP on translation.

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In vitro experimental system for analysis of transcription–translation coupling

Cited by 33 publications

References 38 publications

DksA Guards Elongating RNA Polymerase against Ribosome-Stalling-Induced Arrest

DksA Guards Elongating RNA Polymerase against Ribosome-Stalling-Induced Arrest

Evidence against Translational Repression by the Carboxyltransferase Component of Escherichia coli Acetyl Coenzyme A Carboxylase

Methodology for the analysis of transcription and translation in transcription-coupled-to-translation systems in vitro

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