The Role of the 5′ Untranslated Region of Eukaryotic Messenger RNAs in Translation and Its Investigation Using Antisense Technologies

Pantopoulos, Kostas; Johansson, Hans; Hentze, Matthias W.

doi:10.1016/s0079-6603(08)60856-9

Cited by 17 publications

(13 citation statements)

References 294 publications

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“…A translational regulatory mechanism has been proposed for the E. coli L10 operon that is remarkably similar to the mechanism that we demonstrated for the B. subtilis trp operon (43). In this case, it is thought that binding of the L10-(L12) 4 complex to the untranslated L10 operon leader promotes sequestration of the L10 SD sequence in a stable secondary structure. However, mRNA structural studies have failed to confirm the predicted structural switch (44).…”

Section: Discussionsupporting

confidence: 78%

“…The RNA used in this analysis was synthesized in vitro using the Ambion MEGAscript kit and plasmid pHD15 linearized with BamHI as template. Translation reactions (50 l) contained 72 mM Tris-HCl (pH 7.5), 72 mM NH 4 Cl, 10 mM magnesium acetate, 0.1 mM EDTA (pH 7.5), 2.4 mM dithiothreitol, 2 mM ATP, 0.1 mM GTP, 0.08 mM calcium folinate, 0.2 mM diisopropylfluorophosphate, 20 mM phosphoenolpyruvate, 35 units/ml pyruvate kinase, 1 mM L-tryptophan, 0.1 mM concentration of the remaining amino acids (minus methionine), 4 l of S30 extract (30 mg of total protein), 800 units/ml DNase I, 500 units/ml RNasin, 2 pmol of unlabeled transcript, and 10 Ci of […”

Section: Methodsmentioning

confidence: 99%

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trp RNA-binding Attenuation Protein-mediated Long Distance RNA Refolding Regulates Translation of trpE inBacillus subtilis

Babitzke

1998

Journal of Biological Chemistry

136

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Expression of the trpEDCFBA operon is regulated at both the transcriptional and translational levels by the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis. When cells contain sufficient levels of tryptophan to activate TRAP, the protein binds to trp operon transcripts as they are being synthesized, most often causing transcription termination. However, termination is never 100% efficient, and transcripts that escape termination are subject to translational control. We determined that TRAP-mediated translational control of trpE can occur via a novel RNA conformational switch mechanism. When TRAP binds to the 5-untranslated leader segment of a trp operon read-through transcript, it can disrupt a large secondary structure containing a portion of the TRAP binding target. This promotes refolding of the RNA such that the trpE Shine-Dalgarno sequence, located more than 100 nucleotides downstream from the TRAP binding site, becomes sequestered in a stable RNA hairpin. Results from cell-free translation, ribosome toeprint, and RNA structure mapping experiments demonstrate that formation of this structure reduces TrpE synthesis by blocking ribosome access to the trpE ribosome binding site. The role of the Shine-Dalgarno blocking hairpin in controlling translation of trpE was confirmed by examining the effect of multiple nucleotide substitutions that abolish the structure without altering the Shine-Dalgarno sequence itself. The possibility of protein-mediated RNA refolding as a general mechanism in controlling gene expression is discussed.Studies on the regulation of protein synthesis have shown that the RNA secondary structural features present in the 5Ј-UTR 1 dramatically influence translation initiation in both prokaryotic and eukaryotic organisms (for recent reviews see Refs. 1-7). In prokaryotic mRNAs, a conserved stretch of 4 -6 nucleotides called the Shine-Dalgarno (SD) sequence is usually found 4 -11 nucleotides upstream of the initiation codon. The SD sequence base pairs with the 16 S rRNA present in the 30 S ribosomal subunit and thereby correctly positions the initiation codon in the ribosome (8, 9). Translational control mechanisms have been identified in prokaryotes that involve blocking the SD sequence either by RNA secondary structure (9 -12) or by a bound protein (13-16). In the known translational control mechanisms that occur by formation of SD blocking hairpins, formation of the inhibitory structure is spontaneous and does not require protein factors.Expression of the Bacillus subtilis tryptophan biosynthetic genes is regulated in response to changes in the intracellular level of tryptophan at both the transcriptional and translational levels (for a recent review, see Ref. 17). Six of the seven trp genes are clustered in the trpEDCFBA operon. Transcription of the trp operon is regulated by an attenuation mechanism in which tryptophan-activated trp RNA-binding attenuation protein (TRAP) binds to 11 closely spaced (G/U)AG repeats (seven GAG and four UAG) (18 -25). TRAP binding to the 11 tr...

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Section: Discussionsupporting

confidence: 78%

Section: Methodsmentioning

confidence: 99%

trp RNA-binding Attenuation Protein-mediated Long Distance RNA Refolding Regulates Translation of trpE inBacillus subtilis

Babitzke

1998

Journal of Biological Chemistry

136

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“…RNA molecules can adopt complex structural foldings which prevent the use of antisense oligonucleotides for the artificial regulation of gene expression [1]. Many of these structural elements are implicated in regulatory processes such as the iron responsive element (IRE), which regulates the stability and the translation efficiency of the transferrin receptor and ferritin messages, respectively [2], or the TAR element, which enhances the HIV‐1 transcription upon selective binding to viral and host proteins [3]. Likewise, an RNA hairpin is part of the gag‐pol frameshifting signal of HIV‐1 and acts as a positive modulator of the Pol synthesis [4].…”

Section: Introductionmentioning

confidence: 99%

Binding of oligopyrimidines to the RNA hairpin responsible for the ribosomegag‐polframeshift in HIV‐1

1999

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The 12 bp stem of the RNA hairpin responsible for the gag-pol frameshifting of the ribosomes during translation of the polycistronic HIV-1 mRNA has a pyrimidine-rich 5P P strand and, consequently, a purine-rich 3P P strand. Electrophoretic mobility shift assays have shown that DNA oligopyrimidines, 12 and 20 nucleotides long (but not oligopurines or G,T-containing oligomers), designed to form triplexes actually bind to the double-stranded RNA target. RNase V1 footprinting studies have confirmed the interaction between the hairpin stem and the. RNA and 2P P-O-methyl oligoribonucleotide analogues of the 12-mer oligodeoxypyrimidine as well as 5 propynyl,cytosine, containing the 12-mer oligodeoxypyrimidine, bind more strongly to the RNA target than the unmodified parent DNA oligomer. The complexes formed by the RNA hairpin and either the 12-mer oligodeoxypyrimidine or the 20-mer oligopyrimidine are stable at a neutral pH and in the absence of Mg 2+ but blocked neither the reverse transcription nor cell-free translation of a RNA template in which the gag-pol frameshifting hairpin was inserted at the 5P P end of the luciferase open reading frame.z 1999 Federation of European Biochemical Societies.

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“…Using novel antisense oligonucleotides, he and his colleagues were able to arrest different intermediates along the translation initiation pathway (12,13).…”

mentioning

confidence: 99%

Choosing Industry: Biotech Makes Its Mark

Brownlee¹

2007

ACS Chem. Biol.

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The Role of the 5′ Untranslated Region of Eukaryotic Messenger RNAs in Translation and Its Investigation Using Antisense Technologies

Cited by 17 publications

References 294 publications

trp RNA-binding Attenuation Protein-mediated Long Distance RNA Refolding Regulates Translation of trpE inBacillus subtilis

trp RNA-binding Attenuation Protein-mediated Long Distance RNA Refolding Regulates Translation of trpE inBacillus subtilis

Binding of oligopyrimidines to the RNA hairpin responsible for the ribosomegag‐polframeshift in HIV‐1

Choosing Industry: Biotech Makes Its Mark

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