A Green Fluorescent Protein (GFP)-Based Plasmid System to Study Post-Transcriptional Control of Gene Expression In Vivo

Urban, Johannes; Vogel, Jörg

doi:10.1007/978-1-59745-558-9_22

Cited by 38 publications

(37 citation statements)

References 18 publications

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“…4B, quantified in 4C). We then addressed the discrepancy in EF-P dependent expression of atpA and atpD by employing the previously used pXG10sf translational fusion system to compare translation in wild type and efp mutant Salmonella [11], [27], [28]. The constructs allowed for the constitutive transcription of mRNA bearing full-length atpA or atpD genes with “super-folder” GFP as a C-terminal translational fusion [29].…”

Section: Resultsmentioning

confidence: 99%

EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation

et al. 2014

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Elongation factor P (EF-P) is required for the efficient synthesis of proteins with stretches of consecutive prolines and other motifs that would otherwise lead to ribosome pausing. However, previous reports also demonstrated that levels of most diprolyl-containing proteins are not altered by the deletion of efp. To define the particular sequences that trigger ribosome stalling at diprolyl (PPX) motifs, we used ribosome profiling to monitor global ribosome occupancy in Escherichia coli strains lacking EF-P. Only 2.8% of PPX motifs caused significant ribosomal pausing in the Δefp strain, with up to a 45-fold increase in ribosome density observed at the pausing site. The unexpectedly low fraction of PPX motifs that produce a pause in translation led us to investigate the possible role of sequences upstream of PPX. Our data indicate that EF-P dependent pauses are strongly affected by sequences upstream of the PPX pattern. We found that residues as far as 3 codons upstream of the ribosomal peptidyl-tRNA site had a dramatic effect on whether or not a particular PPX motif triggered a ribosomal pause, while internal Shine Dalgarno sequences upstream of the motif had no effect on EF-P dependent translation efficiency. Increased ribosome occupancy at particular stall sites did not reliably correlate with a decrease in total protein levels, suggesting that in many cases other factors compensate for the potentially deleterious effects of stalling on protein synthesis. These findings indicate that the ability of a given PPX motif to initiate an EF-P-alleviated stall is strongly influenced by its local context, and that other indirect post-transcriptional effects determine the influence of such stalls on protein levels within the cell.

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Section: Resultsmentioning

confidence: 99%

EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation

et al. 2014

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“…To investigate additional regions impacting EF-P dependence, we systematically swapped regions of atpA and atpD using the previously employed pXG10sf plasmid, involving a constitutively active promoter and C-terminally fused superfolder GFP as a reporter (29,(42)(43)(44). We generated serial swaps of increasing length from the 5Ј end of the mRNA transcript to the PPG motif or in the reverse direction (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The pXG10sf translational fusion reporter system was used to measure EF-P-dependent expression in Salmonella strains and has been described previously (29,(42)(43)(44). The plasmid employs a tightly regulated low copy number origin of replication (pSC101) and a constitutively active promoter (PLtet0-1) to minimize variations in transcription.…”

Section: Methodsmentioning

confidence: 99%

Translation Initiation Rate Determines the Impact of Ribosome Stalling on Bacterial Protein Synthesis

Hersch

Elgamal

Katz

et al. 2014

Journal of Biological Chemistry

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Background: Elongation factor P (EF-P) rescues ribosomes stalled at consecutive prolines; however, not all proteins with polyprolines show EF-P-dependent expression. Results: A correlation between translation initiation rate and EF-P dependence is demonstrated. Conclusion: Ribosome stalls lower protein levels only when they are more rate-limiting than initiation. Significance: The results explain why stall motifs do not necessarily affect protein abundance.

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“…If an antibody is not available for the target of interest, a GFP reporter system has been developed that allows rapid fusion of target proteins and detection by fl uorescence or using a commercially available GFP-monoclonal antibody ( 19,20 ) .…”

Section: Functional Analysis Of Tagged Srnasmentioning

confidence: 99%

Use of Aptamer Tagging to Identify In Vivo Protein Binding Partners of Small Regulatory RNAs

Corcoran

Rieder

Podkaminski

et al. 2012

Methods in Molecular Biology

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Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our understanding of the breadth of posttranscriptional control by sRNAs were achieved in a number of pioneering studies that involved immunoprecipitation of a known RNA chaperone, the near-ubiquitous Hfq, followed by sequencing to identify novel putative regulators and targets. To perform the converse experiment, we previously developed a method which uses an aptamer-tagged sRNA to allow purification of in vivo assembled RNA-protein complexes and subsequent identification of bound proteins. We successfully implemented this protocol using the Hfq-associated sRNA, InvR, tagged with a tandem repeat of the commonly used MS2-aptamer. Incorporation of the aptamer had no effect on sRNA stability or activity. InvR-MS2 could be effectively purified along with associated proteins, such as Hfq, using maltose binding protein fused to the MS2 coat protein (MBP-MS2) immobilized on an amylose column. Mass-spectroscopy was also used to identify previously uncharacterized protein partners. These results have been described previously (Said et al., Nucleic Acids Res 37:e133, 2009) and thus the figures presented here are intended solely as an illustrative guide to complement this detailed step-by-step protocol.

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A Green Fluorescent Protein (GFP)-Based Plasmid System to Study Post-Transcriptional Control of Gene Expression In Vivo

Cited by 38 publications

References 18 publications

EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation

EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation

Translation Initiation Rate Determines the Impact of Ribosome Stalling on Bacterial Protein Synthesis

Use of Aptamer Tagging to Identify In Vivo Protein Binding Partners of Small Regulatory RNAs

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