Translational repression: Biological activity of plasmid-encoded bacteriophage T4 RegA protein

Miller, Eric S.; Karam, J.D.; Dawson, Myra; Trojanowska, Maria; Gauss, Peter; Gold, Larry

doi:10.1016/0022-2836(87)90670-x

Cited by 32 publications

(51 citation statements)

References 26 publications

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“…The T4-encoded RegA is one of the few known proteins that regulates the translation of multiple transcripts (26). The mechanism by which the protein discriminates between messages has remained a mystery, as the makeup of the start sites of the affected mRNAs are not statistically unlike those that are unaffected (11).…”

Section: Discussionmentioning

confidence: 99%

Single-stranded RNA Recognition by the Bacteriophage T4 Translational Repressor, RegA

Brown¹,

Brown²,

Kang³

et al. 1997

Journal of Biological Chemistry

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The T4 protein, RegA, is a translational repressor that blocks ribosome binding to multiple T4 messages by interacting with the mRNAs near their respective AUG start codons. Other than the AUG, there are no obvious similarities between the affected mRNAs. High affinity RNA ligands to RegA were isolated using SELEX (systematic evolution of ligands by exponential enrichment). The selected RNAs exhibited the consensus sequence 5-AAAAUUGUUAUGUAA-3. The AUG was invariant, suggesting that it is the primary effector of binding specificity. The UU immediately 5 to the AUG and the upstream poly(A) tract were highly conserved among the selected RNAs. Boundary and footprinting experiments are consistent with the consensus sequence defining the RegA-binding site. Interestingly, chemical modification and nuclease digestion data indicate that the RNA-binding site is single-stranded, as if RegA discriminates between targets based on their primary sequence, not their secondary structure. Minor variations from the consensus at positions other than the universally conserved AUG have little effect on RegA binding, but accumulation of mutations has a profound effect on the interaction. Comparison of the in vivo targets for RegA to the SELEX-generated consensus suggests a repression pattern whereby the translation of individual messages is sequentially halted until the least similarly affected message, the regA gene itself, is repressed.Translational regulation has been shown to be an important means for controlling gene expression in a variety of organisms, both prokaryotic (1, 2) and eukaryotic (3,4). One of the more interesting regulatory mechanisms involves the repression of translation caused by RNA-binding proteins interacting specifically with mRNAs. Many of these translational repressors function by directly competing for mRNA binding with ribosomes, thus decreasing the level of translational initiation (5). Among the well characterized repressors, the bacteriophage T4 translational repressor, RegA, is unusual in that it affects the translation of many independent messages.The expression of at least nine T4 genes is reduced in the latter stages of the phage life cycle by the autoregulated product of the regA gene (6). Transcription of these genes is not altered, and thus RegA-mediated repression occurs post-transcriptionally (7). Genetic analysis and in vitro footprinting indicate that RegA specifically interacts with several of the regulated mRNAs near their translational start sites (8 -10). The presence of RegA alters the binding of the 30 S subunit of Escherichia coli ribosomes to these mRNAs, thus preventing translational initiation (9). Taken together, these data are consistent with RegA altering gene expression in vivo by obstructing ribosome binding to specific mRNAs.Although RegA repression is specific, the mRNA sequences that are bound by the repressor display few similarities. A consensus for the region surrounding the translational start sites of the affected mRNAs is indistinguishable from one generated for all ...

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Section: Discussionmentioning

confidence: 99%

Single-stranded RNA Recognition by the Bacteriophage T4 Translational Repressor, RegA

Brown¹,

Brown²,

Kang³

et al. 1997

Journal of Biological Chemistry

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“…All the repressor proteins appear to function through occlusion of a portion of the mRNA sequence recognized by the ribosome during translation initiation (2,7,30). The bacteriophage T4 genome encodes at least three translational repressors: gene 32 single-stranded DNA-binding protein (15); gene 43 DNA polymerase (1); and RegA, whose only known function is translational repression (12,16,34). Among translational repressors, RegA uniquely represses several mRNAs, many of which encode enzymes for T4 DNA precursor biosynthesis or DNA replication (33).…”

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confidence: 99%

“…Some of the 12 or more regulated mRNAs are polycistronic transcripts with multiple RegA-binding sites; others are separate unlinked transcripts. RegA therefore has been described as a global translational regulatory protein (16). The RegA-binding sites on the rIIB and gene 44 mRNAs have been analyzed genetically and biochemically, but further characterization of the operator sequences on all the RegA-sensitive mRNAs is needed to appreciate fully the hierarchical site recognition demonstrated by the protein (12,16,32,34).…”

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confidence: 99%

“…RegA therefore has been described as a global translational regulatory protein (16). The RegA-binding sites on the rIIB and gene 44 mRNAs have been analyzed genetically and biochemically, but further characterization of the operator sequences on all the RegA-sensitive mRNAs is needed to appreciate fully the hierarchical site recognition demonstrated by the protein (12,16,32,34). Although the RegA protein has been purified (17) and its inferred amino acid sequence is known (28), nothing is known of functionally important regions of RegA or of amino acids involved in RNA binding by the repressor.…”

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Sequence analysis of conserved regA and variable orf43.1 genes in T4-like bacteriophages

Miller

Jozwik

1990

J Bacteriol

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Bacteriophage T4 RegA protein is a translational repressor of several phage mRNAs. In the T4-related phages examined, regA nucleotide sequences are highly conserved and the inferred amino acid sequences are identical. The exceptional phage, RB69, did not produce a RegA protein reproducibly identifiable by Western blots (immunoblots) nor did it produce mRNA that hybridized to T4 regA primers. Nucleotide sequences of either 223 or 250 base pairs were identified immediately 3' to regA in RB18 and RB51 that were absent in T-even phages. Open reading frames' in these regions, designated orf43.lRBl8 and orf43.lRB59l potentially encode related proteins of 8.5 and 9.2 kilodaltons, respectively. orf43.1 sequences, detected in 13 of 27 RB bacteriophage chromosomes analyzed by polymerase chain reaction, are either RB18-or RB51-like and have flanking repeat sequences that may promote orf43.1 deletion.

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“…In addition, plasmid-encoded repressor proteins have been shown to down-regulate expression (31,34). Berkhout and Jeang found that a prokaryotic RNA-binding protein is able to down-regulate human immunodeficiency virus type 1 gene expression (7).…”

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Evolution of Bacteriophage in Continuous Culture: a Model System To Test Antiviral Gene Therapies for the Emergence of Phage Escape Mutants

Lindemann

Klug²,

Schwienhorst

2002

J Virol

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The emergence of viral escape mutants is usually a highly undesirable phenomenon. This phenomenon is frequently observed in antiviral drug applications for the treatment of viral infections and can undermine long-term therapeutic success. Here, we propose a strategy for evaluating a given antiviral approach in terms of its potential to provoke the appearance of resistant virus mutants. By use of Q␤ RNA phage as a model system, the effect of an antiviral gene therapy, i.e., a virus-specific repressor protein expressed by a recombinant Escherichia coli host, was studied over the course of more than 100 generations. In 13 experiments carried out in parallel, 12 phage populations became resistant and 1 became extinct. Sequence analysis revealed that only two distinct phage mutants emerged in the 12 surviving phage populations. For both escape mutants, sequence variations located in the repressor binding site of the viral genomic RNA, which decrease affinity for the repressor protein, conferred resistance to translational repression. The results clearly suggest the feasibility of the proposed strategy for the evaluation of antiviral approaches in terms of their potential to allow resistant mutants to appear. In addition, the strategy proved to be a valuable tool for observing virus-specific molecular targets under the impact of antiviral drugs.Ideally, antiviral strategies should be highly virus specific and should not affect the host organism. In addition, they should exhibit a minimal potential to allow viral escape mutants to appear. In this paper, we propose a general strategy to evaluate a given antiviral approach with regard to resistance phenomena that may occur due to the emergence of escape mutants. Utilizing RNA phage Q␤ as a model system, we investigated the potential of virus-specific translational repressor proteins to suppress viral propagation. Among the mechanisms that control gene expression, translational regulation is certainly one of the most specific and therefore should be a valuable target mechanism for antiviral strategies.First observed in the RNA phages (29), translational repression has since been discovered in many bacteriophages (47), prokaryotes (11,16,45), and eukaryotes (46) as well as in several mammalian viruses (14,17,35,44). Within the closely related RNA phage species R17, MS2, F2, fr, and Q␤, two proteins, namely, the coat protein and the replicase, are translational regulators of each other. Shortly after single-stranded viral RNA enters the host cell, replicase protein is translated from the proximal end of the phage genome, leading to high levels of replicated phage RNA.However, it has been shown that replication cannot occur when phage RNA is being translated (25,47). Therefore, strict translational regulation of the remaining cistrons, namely, A1/ coat protein and A2, from the distal portion of the Q␤ RNA genome is essential for the replication. Hence, blocking the translation of the coat protein, which is produced abundantly during the late phase of the viral infection cycl...

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Translational repression: Biological activity of plasmid-encoded bacteriophage T4 RegA protein

Cited by 32 publications

References 26 publications

Single-stranded RNA Recognition by the Bacteriophage T4 Translational Repressor, RegA

Single-stranded RNA Recognition by the Bacteriophage T4 Translational Repressor, RegA

Sequence analysis of conserved regA and variable orf43.1 genes in T4-like bacteriophages

Evolution of Bacteriophage in Continuous Culture: a Model System To Test Antiviral Gene Therapies for the Emergence of Phage Escape Mutants

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