Interdependence of translation, transcription and mRNA degradation in the lacZ gene

Yarchuk, O.; Jacques, Nathalie; Guillerez, Jean; Dreyfus, Marc

doi:10.1016/0022-2836(92)90617-s

Cited by 170 publications

(159 citation statements)

References 54 publications

Supporting

Mentioning

153

Contrasting

Unclassified

Order By: Relevance

“…1 RNA isolation and analysis Samples of the same cultures used for protein assays were rapidly mixed with an equal volume of boiling lysis buffer (5% SDS, 1.5 M sodium acetate, 12 mM EDTA, pH 7.0), and total RNA was extracted as in (21). RNA samples (5 or 20 ptg) were electrophoresed on 2% agarose-formaldehyde gels, blotted and UV cross-linked onto a nylon membrane (Hybond-N+, Amersham) as in (18). For the visualisation of 5S rRNA and tRNAArg5, the blots were probed and washed as in (22).…”

Section: Methodsmentioning

confidence: 99%

“…4, the 845 bp BgllIBstBI fragment from the pNEO plasmid was used as a probe after being 32p labelled with the BRL multiprime labelling kit. The membrane was processed as in (18).…”

Section: Methodsmentioning

confidence: 99%

“…All three are preceded by the repressor binding site and followed by the tRNA gene. Due to construction constraints, the lacZ construct differs from others in two respects: (i) the lacZ gene is not translated from its own RBS but from that of the lamB gene, which is nearly equivalent in efficiency (18); and (ii) it is followed by a truncated version of the lacY gene, making this construct tri-rather than bicistronic (Fig. 1).…”

Section: In Vitro Transcriptionmentioning

confidence: 99%

See 2 more Smart Citations

The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient inEscherichia colibut its transcripts are poorly expressed

1994

Self Cite

View full text Add to dashboard Cite

In gene expression studies, promoters are often fused to a protein-encoding reporter gene, the expression of which is then taken as an indirect measure of their strength. Here, we advocate the use of a tRNA reporter for the direct quantification of promoter strength. Using this method, we have studied the bacteriophage T7 gene 10 promoter in an E.coli strain that produces saturating amounts of T7 RNA polymerase. At 370C in aminoacid-glycerol medium, we show that this promoter ranks amongst the strongest known, directing ca 1.1 transcription events per second, 2.2-fold more than the promoters for rRNA operons, or 15-fold more than the induced lac promoter. Surprisingly, compared to the lac promoter, the T7 promoter is far less efficient in driving the expression of protein-encoding genes such as cat, neo or lacZ. Therefore, the polypeptide yield per transcript is lower when the T7 RNA polymerase is used instead of the E.coli RNA polymerase. The former enzyme travels faster than the translating ribosomes, and we suggest that this desynchronization lowers the polypeptide yield per transcript. INTRODUCTIONBacteriophages T3, T7 and SP6 encode closely related RNA polymerases which transcribe the viral genome from highly specific promoters, during the late phase of infection (1). The prototype of the group, the T7 RNA polymerase, can be stably expressed in E. coli, and genes that are fused to a T7 late promoter can be expressed to high levels when introduced in such hosts (2, 3). In spite of this practical bearing, relatively little is known about the in vivo behaviour of these RNA polymerases. Indirect evidence suggests that the T7 RNA polymerase interacts very efficiently with its promoter in E. coli (4), whereas in vitro the interaction appears relatively weak (1,5). The enzyme has a very high turnover for the polymerisation of ribonucleotides, both in vitro (6) and in vivo (7). As a result, in E. coli it runs far ahead of the ribosomes which translate its nascent transcript, a situation

show abstract

Section: Methodsmentioning

confidence: 99%

“…4, the 845 bp BgllIBstBI fragment from the pNEO plasmid was used as a probe after being 32p labelled with the BRL multiprime labelling kit. The membrane was processed as in (18).…”

Section: Methodsmentioning

confidence: 99%

Section: In Vitro Transcriptionmentioning

confidence: 99%

See 1 more Smart Citation

The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient inEscherichia colibut its transcripts are poorly expressed

1994

Self Cite

View full text Add to dashboard Cite

show abstract

“…For example, several previous studies in Escherichia coli and Bacillus subtilis have shown that for particular model transcripts, increased ribosome binding or occupancy may enhance mRNA stability (36)(37)(38)(39)(40)(41)(42). Such studies have been hard to interpret due to the complex interactions between the ribosome, RNA degradation machinery, and transcript.…”

Section: Resultsmentioning

confidence: 99%

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Kosuri

Goodman

Cambray

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

415

453

View full text Add to dashboard Cite

The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.next-generation sequencing | synthetic biology | systems biology O rganisms can be engineered to produce chemical, material, fuel, and medical products that are often superior to nonbiological alternatives (1). Biotechnologists have sought to discover, improve, and industrialize such products through the use of recombinant DNA technologies (2, 3). In recent years, these efforts have increased in complexity from expressing a few genes at once to optimizing multicomponent circuits and pathways (4-7). To attain desired systems-level function reliably, careful and time-consuming optimization of individual components is required (8-11).To mitigate this slow trial-and-error optimization, two dominant approaches have taken hold. The first approach seeks to predict expression levels by elucidating the biophysical relationships between sequence and function. For example, several groups have modified promoters (12, 13) and ribosome binding sites (RBSs) (14-16) to see how small sequence changes affect transcription or translation. Such studies are fundamentally challenging due to the vastness of sequence space. In addition, because these approaches mostly look at either transcription or translation individually, they are rarely able to investigate interactions between these processes.The second approach uses combinations of individually characterized elements to attain desired expression without directly considering their DNA sequences (17-25). Current efforts have focused on approaches to limit the number of time-consuming steps required to characterize potential interactions and on identifying existing or engineered elements that act predictably when used in combination (26-28). However, these studies still suggest there are enough idiosyncratic interactions and context effects that it will be necessary to construct and measure many variants of a circuit to achieve desired function (29). For larger circuits, such approaches are necessarily limited in scope due to the difficulty in measuring large numbers of combinations (26, 27...

show abstract

“…Another way by which ribosomes could affect degradation is by melting out, during translation, secondary structures that may be determinants recognized by RNases. Among the RNases that have been shown to be sensitive to ribosomes is RNase E, whose efficacy of action is inversely related to the rate of translation (35).…”

Section: Cofactors In Rna Degradationmentioning

confidence: 99%

Degradation of mRNA in Escherichia coli

Jain

2002

IUBMB Life

View full text Add to dashboard Cite

SummaryDegradation of messenger RNAs (mRNAs) is a universal process that occurs in every cell and has important implications for nucleotide metabolism and gene expression. One organism in which mRNA degradation has been thoroughly studied is the bacterium Escherichia coli (E. coli). In this review I describe what is presently known about the different processes involved in the conversion of mRNAs from high molecular weight species to mononucleotides in E. coli. The ribonucleases and accessory factors involved in mRNA degradation, and features on mRNAs that make them resistant or sensitive to degradation will also be described. At the conclusion of this review, some of the anticipated directions of future research on this topic will be discussed.

show abstract

Interdependence of translation, transcription and mRNA degradation in the lacZ gene

Cited by 170 publications

References 54 publications

The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient inEscherichia colibut its transcripts are poorly expressed

The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient inEscherichia colibut its transcripts are poorly expressed

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Degradation of mRNA in Escherichia coli

Contact Info

Product

Resources

About