Complex formation by positive and negative translational regulators of GCN4.

Cigan, A. Mark; Foiani, Marco; Hannig, Ernest M.; Hinnebusch, Alan G.

doi:10.1128/mcb.11.6.3217

Cited by 117 publications

(115 citation statements)

References 58 publications

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“…Possibilities for the role of these 5Ј untranslated regions include control over translocation of the mRNA within the cell, translational control, or transcriptional control. Although no upstream, in-frame translation start sites were seen, it is possible that short out-of-frame translation products could be used to regulate protein synthesis, as has been observed for yeast GCN4 (33,34). In addition, it has been observed that ORG mRNA is found both at the dendrite and at the axon (28).…”

Section: Discussionmentioning

confidence: 93%

Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse β-globin gene clusters

Bulger

Bender

Doorninck

et al. 2000

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

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By sequencing regions flanking the ␤-globin gene complex in mouse (Hbbc) and human (HBBC), we have shown that the ␤-globin gene cluster is surrounded by a larger cluster of olfactory receptor genes (ORGs). To facilitate sequence comparisons and to investigate the regulation of ORG expression, we have mapped 5 sequences of mRNA from olfactory epithelium encoding ␤-globinproximal ORGs. We have found that several of these genes contain multiple noncoding exons that can be alternatively spliced. Surprisingly, the only common motifs found in the promoters of these genes are a ''TATA'' box and a purine-rich motif. Sequence comparisons between human and mouse reveal that most of the conserved regions are confined to the coding regions and transcription units of the genes themselves, but a few blocks of conserved sequence also are found outside of ORG transcription units. The possible influence of ␤-globin regulatory sequences on ORG expression in olfactory epithelium was tested in mice containing a deletion of the endogenous ␤-globin locus control region, but no change in expression of the neighboring ORGs was detected. We evaluate the implications of these results for possible mechanisms of regulation of ORG transcription.O lfactory receptors are a family of seven-transmembrane G protein-coupled receptors expressed in sensory neurons of nasal epithelium, where they bind to odorant epitopes and transduce this primary signal into membrane potential (1-3). These molecules are encoded by a family of up to 1,000 genes in mouse and human, which are grouped into discrete clusters at different chromosomal locations (4-7). Production of a given olfactory receptor is restricted to one of four spatially defined domains within the olfactory epithelium (8,9). Despite the large size of this gene family, each olfactory neuron appears to express only a single olfactory receptor gene (ORG) (10, 11) in an allele-specific manner (12). The mechanisms that underlie any of these regulatory decisions-expression in olfactory neurons, zonal specificity, one receptor per neuron, or allelic exclusionare undefined. In addition, expression of some ORGs has been documented in nonolfactory tissues (13-17), but the significance of such expression is also unknown.The characterization of DNA sequences required for proper gene expression, such as promoters and enhancers, represents a basic approach to such questions. Functional studies of putative ORG regulatory elements, however, are hindered by the lack of a viable cell culture model for olfactory neurons. Although analysis of transgenes in mice provides a suitable model system, the production of mouse lines is time-consuming and expensive. Thus, genomic approaches are particularly relevant to the identification of ORG regulatory sequences, in part to guide the selection of regions to be examined by functional studies in transgenic mice. Candidates for such regions can be recognized as noncoding sequences that are conserved between species (18).Previously, we reported that ORGs surround the complex o...

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Section: Discussionmentioning

confidence: 93%

Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse β-globin gene clusters

Bulger

Bender

Doorninck

et al. 2000

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

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“…Addition of eIF-2 increased initiation on the upstream AUG. The conclusion was that the increased availability of active eIF-2 increased the concentration of eIF-2-Met-tRNA-GTP-40S complex and therefore increased initiation on the upstream AUG. A very similar mechanism regulates yeast GCN4 mRNA translation (Cigan et al, 1989(Cigan et al, , 1991. Initiation codon selection by eIF-2 has now been shown for capdependent initiation (Dasso et al, 1990; this study), for initiation by leaky scanning and termination-reinitiation (Cigan et al, 1988) and for internal initiation on FMDV-CAT RNA (Figs 3 6).…”

Section: Discussionmentioning

confidence: 99%

“…In the absence of eIF-2 the ribosome is unable to initiate at the nearby AUG and moves downstream to AUGL. o, a situation very similar to initiation on the yeast GCN4 mRNA (Cigan et al, 1989(Cigan et al, , 1991. During movement towards AUGLb, eIF-2 binds to 40S, unless the activity or amount of eIF-2 is too low, resulting in abortive initiation.…”

Section: Discussionmentioning

confidence: 99%

Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA

Thomas

Rijnbrand

Voorma

1996

Journal of General Virology

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Foot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5' untranslated region (5'UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5'UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products. After in vitro transcription with T7 RNA polymerase and translation in rabbit reticulocyte lysate, the two CAT products, resulting from initiation from the two initiation codons, were quantified. The downstream initiator AUG (AUGLb) was selected more efficiently in the wild-type 5'UTR. In truncated RNA, the upstream initiation site (AUGL~b) was more efficiently utilized than in the wild-type 5'UTR. Protein synthesis initiation factors were added to translation assays to determine whether these factors influenced initiation site selection. Addition of eIF-2 and of eIF-2B changed the selection process for both types of RNA. These factors induced a 2.5-fold higher usage of the upstream AUGL~ b for wildtype and 5'UTR-truncated RNA. A change in mRNA concentration also induced a change in the usage of initiation codons; however, the effect of elF-2 was measured over a broad range of mRNA concentrations. In conclusion, elF-2 mediates the recognition of the initiation codon during both cap-dependent and internal ribosome entry site-dependent initiation.

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“…Cells were grown in yeast nitrogen base medium at 30ЊC to 5 ϫ 10 6 to 8 ϫ 10 6 cells per ml, washed twice with ice-cold H 2 O, resuspended in ice-cold lysis buffer consisting of 40 mM PIPES [piperazine-N,NЈ-bis(2-ethanesulfonic acid)] (pH 6.0)-100 mM NaCl-1 mM dithiothreitol containing protease inhibitors (11), and lysed by agitation with glass beads. The cell extract was resolved and immunoblotted as described previously (11) with anti-Prt1p primary antibody against Prt1p (11) or eIF-2␣ (a gift from Alan Hinnebusch).…”

Section: Construction Of Mutations In Vitromentioning

confidence: 99%

Mutational Analysis of the Prt1 Protein Subunit of Yeast Translation Initiation Factor 3

Evans

Rasmussen

Hanic-Joyce

et al. 1995

Molecular and Cellular Biology

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The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5 end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.Mutations in the PRT1 gene of the yeast Saccharomyces cerevisiae, as exemplified by the temperature-sensitive prt1-1 mutant allele, cause a conditional impairment of translation initiation in vivo (26, 27; see also reference 18); in vitro, prt1-1 mutant cell extracts are defective for the interaction of the eIF-2 GTP Met-tRNA i ternary complex with the 40S ribosomal subunit (16), suggesting that the PRT1 polypeptide (Prt1p) may be a component of the yeast translation initiation factor 3 (eIF-3) complex (37, 38). Recently, this suggestion has been validated by the identification of Prt1p in purified yeast .Mutant forms of the PRT1 gene have been found several times by selection for temperature-sensitive growth mutants (26,27,58) and also through searches for more-specific effects. The prt1-63 allele (formerly cdc63 [22]) was obtained by selection for mutants conditionally unable to perform the G 1 cell cycle regulatory step (2), whereas the prt1-26 allele (formerly dna26 [15]) was identified in a mutant defective for DNA synthesis (13). The identification of prt1 mutant alleles by the latter criteria can be understood from the effects of prt1 mutations on the cell cycle. At appropriate restrictive temperatures, all temperature-sensitive prt1 mutations can cause regulated cell cycle blockage (23), and the DNA synthesis impairment caused by the prt1-26 mutation was shown to be an indirect consequence of a concerted arrest of mutant cells in the G 1 phase of the cell cycle (15). Thus, prt1 mutant alleles can allow sufficient protein synthesis for completion of an ongoing cell cycle (7) while blocking initiation of the next cell cycle and DNA replication.Prt1p has been characterized by analysis of the cloned PRT1 gene (24). In this report, we extend the characterization of Prt1p by showing that the open reading frame (ORF) of PRT1 does not accurately predict Prt1p and by identi...

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Complex formation by positive and negative translational regulators of GCN4.

Cited by 117 publications

References 58 publications

Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse β-globin gene clusters

Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse β-globin gene clusters

Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA

Mutational Analysis of the Prt1 Protein Subunit of Yeast Translation Initiation Factor 3

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