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1.
Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate translation, is the actual start codon in mouse TGR. The use of this codon relies on the Kozak consensus sequence and ribosome-scanning mechanism. However, CUG serves as an inefficient start codon that allows downstream initiation, thus generating two isoforms of the enzyme in vivo and in vitro. The use of CUG evolved in mammalian TGRs, and in some of these organisms, GUG is used instead. The newly discovered longer TGR form shows cytosolic localization in cultured cells and is expressed in spermatids in mouse testes. This study shows that CUG codon is used as an inefficient start codon to generate protein isoforms in mouse.  相似文献   

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Nucleotide sequences around the boundaries of all open reading frames in the Escherichia coli whole genome were analyzed. Characteristic base biases were observed after the initiation codon and before the termination codon. We examined the effect of the base sequence after the initiation codon on the translation efficiency, by introducing mutations after the initiation codon of the E. coli dihydrofolate reductase (DHFR) gene, considering codon and base biases, and using in vitro and in vivo translation systems. In both assay systems, the two most frequent second codons, AAA and AAU, enhanced the translation efficiency compared with the wild type, whereas the effects of lower frequency codons were not significant. Experiments using 16S rRNA variants with mutations in the putative complementary sequence to the region downstream of the initiation codon showed that the translation efficiency of none of the DHFR mutants was affected. These results demonstrate that the statistically most frequent sequences for the second codon enhance translation efficiency, and this effect seems to be independent of base pairing between mRNA and 16S rRNA.  相似文献   

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To examine normal and aberrant translation initiation in Saccharomyces cerevisiae mitochondria, we fused the synthetic mitochondrial reporter gene ARG8m to codon 91 of the COX2 coding sequence and inserted the chimeric gene into mitochondrial DNA (mtDNA). Translation of the cox2(1-91)::ARG8m mRNA yielded a fusion protein precursor that was processed to yield wild-type Arg8p. Thus mitochondrial translation could be monitored by the ability of mutant chimeric genes to complement a nuclear arg8 mutation. As expected, translation of the cox2(1-91)::ARG8m mRNA was dependent on the COX2 mRNA-specific activator PET111. We tested the ability of six triplets to function as initiation codons in both the cox2(1-91)::ARG8m reporter mRNA and the otherwise wild-type COX2 mRNA. Substitution of AUC, CCC or AAA for the initiation codon abolished detectable translation of both mRNAs, even when PET111 activity was increased. The failure of these mutant cox2(1-91)::ARG8m genes to yield Arg8p demonstrates that initiation at downstream AUG codons, such as COX2 codon 14, does not occur even when normal initiation is blocked. Three mutant triplets at the site of the initiation codon supported detectable translation, with efficiencies decreasing in the order GUG, AUU, AUA. Increased PET111 activity enhanced initiation at AUU and AUA codons. Comparisons of expression, at the level of accumulated product, of cox2(1-91)::ARG8m and COX2 carrying these mutant initiation codons revealed that very low-efficiency translation can provide enough Cox2p to sustain significant respiratory growth, presumably because Cox2p is efficiently assembled into stable cytochrome oxidase complexes.  相似文献   

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The use of several translation initiation codons in a single mRNA, by expressing several proteins from a single gene, contributes to the generation of protein diversity. A small, yet growing, number of mammalian mRNAs initiate translation from a non-AUG codon, in addition to initiating at a downstream in-frame AUG codon. Translation initiation on such mRNAs results in the synthesis of proteins harbouring different amino terminal domains potentially conferring on these isoforms distinct functions. Use of non-AUG codons appears to be governed by several features, including the sequence context and the secondary structure surrounding the codon. Selection of the downstream initiation codon can occur by leaky scanning of the 43S ribosomal subunit, internal entry of ribosome or ribosomal shunting. The biological significance of non-AUG alternative initiation is demonstrated by the different subcellular localisations and/or distinct biological functions of the isoforms translated from the single mRNA as illustrated by the two main angiogenic factor genes encoding the fibroblast growth factor 2 (FGF2) and the vascular endothelial growth factor (VEGF). Consequently, the regulation of alternative initiation of translation might have a crucial role for the biological function of the gene product.  相似文献   

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A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated. Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene. pSAT1 contained an open reading frame with ca. 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2. Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X5-HF. The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs. Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli. Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown). We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2. N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2. Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.  相似文献   

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Human T-cell lymphotropic virus type I (HTLV-I) double-spliced mRNA exhibits two GUG and two CUG codons upstream to, and in frame with, the sequences encoding Rex and Tax regulatory proteins, respectively. To verify whether these GUG and CUG codons could be used as additional initiation codons of translation, two chimeric constructs were built for directing the synthesis of either Rex–CAT or Tax–CAT fusion proteins. In both cases, the CAT reporter sequence was inserted after the Tax AUG codon and in frame with either the Rex or Tax AUG codon. Under transient expression of these constructs, other proteins of higher molecular mass were synthesized in addition to the expected Rex–CAT and Tax–CAT proteins. The potential non-AUG initiation codons were exchanged for either an AUG codon or a non-initiation codon. This allowed us to demonstrate that the two GUG codons in frame with the Rex coding sequence, and only the second CUG in frame with the Tax coding sequence, were used as additional initiation codons. In HTLV-I infected cells, two Rex and one Tax additional proteins were detected that exhibited molecular mass compatible with the use of the two GUG and the second CUG as additional initiation codons of translation. Comparison of the HTLV-I proviral DNA sequence with that of other HTLV-related retroviruses revealed a striking conservation of the three non-AUG initiation codons, strongly suggesting their use for the synthesis of additional Rex and Tax proteins.  相似文献   

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Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system, which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. In mammals, one gene encodes two forms of the reductase, one targeted to the cytosol and the other to mitochondria. The cytosolic form displays faster mobility than the mitochondrial form, suggesting a lower molecular weight for the former. The apparent size difference and targeting to two cellular compartments had been proposed to result from differential splicing of mRNA. We now show that differential targeting is effected by use of two initiation sites, one of which includes a mitochondrial targeting sequence, whereas the other does not. We also demonstrate that the mass of the cytosolic form is not less than that of the mitochondrial form; the faster mobility of cytosolic form is due to its myristoylation. Lipidation of methionine sulfoxide reductase A occurs in the mouse, in transfected tissue culture cells, and even in a cell-free protein synthesis system. The physiologic role of myristoylation of MsrA remains to be elucidated.  相似文献   

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Stenström CM  Jin H  Major LL  Tate WP  Isaksson LA 《Gene》2001,263(1-2):273-284
The codon that follows the AUG initiation triplet (+2 codon) affects gene expression in Escherichia coli. We have extended this analysis using two model genes lacking any apparent Shine-Dalgarno sequence. Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained. The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability. However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA. High adenine content of the +2 codon was associated with high gene expression. Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon. Analysis of the 3540 E. coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes. Codons that are associated with low gene expression are under-represented. The data suggest that evolution has favored codons at the +2 position that give high translation initiation.  相似文献   

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In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene ( tnp ), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se , was not increased. Rather, these mutations appear to increase expression of the tnp'–'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo : Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1–3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification. Importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3–5-fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo , consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.  相似文献   

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Simple sequence repeats located within reading frames mediate phase-variable ON/OFF switches in gene expression by generating frameshifts. Multiple translation initiation codons in different reading frames are found upstream of most Haemophilus influenzae tetranucleotide repeat tracts, raising the possibility of multiple active reading frames and more than two levels of gene expression for these loci. Phase variation between three levels of gene expression (strong, weak, and none) was observed when lic2A was fused to a lacZ reporter gene. The lic2A 5' CAAT repeat tract is preceded by four 5' ATG codons (x, y, z1, and z2) in two reading frames. Each of these initiation codons was inactivated by site-directed mutagenesis. Strong expression from frame 1 was associated with x but not y. Weak expression from frame 2 was mainly dependent on the z2 codon, and there was no expression from frame 3. Using monoclonal antibodies specific for a digalactoside epitope of lipopolysaccharide whose synthesis requires Lic2A, two levels (strong and undetectable) of antibody reactivity were detected, suggesting that weak expression of lic2A is not discernible at the phenotypic level. Inactivation of the x initiation codon resulted in loss of strong expression of the digalactoside epitope and elevated killing by human serum. The failure to detect more than two phenotypes for lic2A, despite clear evidence of weak expression from the z1/z2 initiation codons, leaves open the question of whether or not multiple initiation codons are associated with more complex patterns of phenotypic variation rather than classical phase-variable switching between two phenotypes.  相似文献   

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lacZ translation initiation mutations   总被引:32,自引:0,他引:32  
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The quantitative levels of initiation of protein synthesis at codons other than AUG were determined with a CYC7-lacZ fused gene in the yeast Saccharomyces cerevisiae. AUG was the only codon which efficiently initiated translation, although some non-AUG codons allowed initiation at very low efficiency, below 1% of the normal level. Since translation initiates at codons other than AUG in at least two wild-type genes from eucaryotes, other factors presumably play a role in enhancing the activity of non-AUG codons.  相似文献   

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