Understanding the Formation of the MCT‐1:DenR Complex, a Translational Enhancer for Lymphoma Survival

ABSTRACT The fepB gene encodes a periplasmic binding protein that is essential for the uptake of ferric enterobactin by Escherichia coli. Its transcription is regulated in response to iron levels by the Fur repressor. The fepB transcript includes a 217-nucleotide leader sequence with several features suggestive of posttranscriptional regulation. To investigate the fepB leader for its contribution to fepB expression, defined deletions and substitution mutations in the leader were characterized using fepB-phoA translational fusions. The fepB leader was found to be necessary for maximal fepB expression, primarily due to the influence of an AU-rich translational enhancer (TE) located 5′ to the Shine-Dalgarno sequence. Deletions or substitutions within the TE sequence decreased fepB-phoA expression fivefold. RNase protection and in vitro transcription-translation assays demonstrated that the TE augmented translational efficiency, as well as RNA levels. Moreover, primer extension inhibition assays showed that the TE increases ribosome binding. In contrast to the enhancing effect of the TE, the natural fepB GUG start codon decreased ribosome binding and reduced fepB expression 2.5-fold compared with the results obtained with leaders bearing an AUG initiation codon. Thus, the TE-GUG organization in fepB results in an intermediate level of expression compared to the level with AUG, with or without the TE. Furthermore, we found that the TE-GUG sequence is conserved among the eight gram-negative strains examined that have fepB genes, suggesting that this organization may provide a selective advantage.

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The 5′-untranslated region of the Oryza sativa alcohol dehydrogenase gene functions as a translational enhancer in monocotyledonous plant cells

Journal of Bioscience and Bioengineering ◽

10.1263/jbb.105.300 ◽

2008 ◽

Vol 105 (3) ◽

pp. 300-302 ◽

Cited By ~ 47

Author(s):

Tadatoshi Sugio ◽

Junko Satoh ◽

Hideyuki Matsuura ◽

Atsuhiko Shinmyo ◽

Ko Kato

Keyword(s):

Oryza Sativa ◽

Alcohol Dehydrogenase ◽

Untranslated Region ◽

Plant Cells ◽

Monocotyledonous Plant ◽

Translational Enhancer ◽

Dehydrogenase Gene ◽

Gene Functions

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An adenosyl-cobalamin (coenzyme-B12)-repressed translational enhancer in the cob mRNA of Salmonella typhimurium

Molecular Microbiology ◽

10.1046/j.1365-2958.2001.02346.x ◽

2001 ◽

Vol 39 (6) ◽

pp. 1585-1594 ◽

Cited By ~ 42

Author(s):

Solveig Ravnum ◽

Dan I. Andersson

Keyword(s):

Salmonella Typhimurium ◽

Coenzyme B12 ◽

Translational Enhancer

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Translation initiation from sequence variants of the bacteriophage T7 g10RBS in Escherichia coli and Agrobacterium fabrum

Molecular Biology Reports ◽

10.1007/s11033-021-06891-z ◽

2021 ◽

Author(s):

Alex B. Benedict ◽

Joshua D. Chamberlain ◽

Diana G. Calvopina ◽

Joel S. Griffitts

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Bacteriophage T7 ◽

Translation Efficiency ◽

Nucleotide Substitutions ◽

Reporter Protein ◽

E Coli ◽

Translational Enhancer ◽

Multiple Promoter ◽

Derivatives Of

Abstract Background The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine–Dalgarno (SD) sequence augmented by an upstream translational “enhancer” (Enh) element, supporting protein production at many times the level seen with simple synthetic SD-containing sequences. The objective of this study was to dissect the g10RBS to identify simpler derivatives that exhibit much of the original translation efficiency. Methods and results Twenty derivatives of g10RBS were tested using multiple promoter/reporter gene contexts. We have identified one derivative (which we call “CON_G”) that maintains 100% activity in E. coli and is 33% shorter. Further minimization of CON_G results in variants that lose only modest amounts of activity. Certain nucleotide substitutions in the spacer region between the SD sequence and initiation codon show strong decreases in translation. When testing these 20 derivatives in the alphaproteobacterium Agrobacterium fabrum, most supported strong reporter protein expression that was not dependent on the Enh. Conclusions The g10RBS derivatives tested in this study display a range of observed activity, including a minimized version (CON_G) that retains 100% activity in E. coli while being 33% shorter. This high activity is evident in two different promoter/reporter sequence contexts. The array of RBS sequences presented here may be useful to researchers in need of fine-tuned expression of recombinant proteins of interest.

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Uncoupled Expression of p33 and p92 Permits Amplification of Tomato Bushy Stunt Virus RNAs

Journal of Virology ◽

10.1128/jvi.72.7.5845-5851.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5845-5851 ◽

Cited By ~ 74

Author(s):

Sara K. Oster ◽

Baodong Wu ◽

K. Andrew White

Keyword(s):

Viral Genome ◽

Stop Codon ◽

Rna Virus ◽

Rna Replication ◽

Tomato Bushy Stunt Virus ◽

Viral Rna ◽

Rna Amplification ◽

Viral Rnas ◽

Reading Frame ◽

Translational Enhancer

ABSTRACT Tomato bushy stunt virus (TBSV) is a plus-sense RNA virus which encodes a 33-kDa protein in its 5′-most open reading frame (ORF). Readthrough of the amber stop codon of the p33 ORF results in the production of a 92-kDa fusion protein. Both of these products are expressed directly from the viral genome and are suspected to be involved in viral RNA replication. We have investigated further the roles of these proteins in the amplification of viral RNAs by using a complementation system in which p33 and p92 are expressed from different viral RNAs. Our results indicate that (i) both of these proteins are necessary for viral RNA amplification; (ii) translation of these proteins can be uncoupled while maintaining amplification of viral RNAs; (iii) if compatibility requirements exist between p33 and p92, they are not exceptionally strict; and (iv) the C-terminal ∼6% of p33 is necessary for its functional activity. Interestingly, no complementation was observed when a p33-encoding replicon containing a deletion of a 3′-located segment, region 3.5, was tested. However, when 5′-capped transcripts of the same replicon were analyzed, complementation allowing for RNA amplification was observed. This ability to compensate functionally for the absence of region 3.5 by the addition of a 5′ cap suggests that this RNA segment may act as a translational enhancer for the expression of virally encoded products.

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