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At least 19 recordsLinked to original sources

PubMed1990

CUG as a mutant start codon for cat-86 and xylE in Bacillus subtilis.

The cat-86 gene specifies chloramphenicol acetyltransferase (CAT). The cat-86 start codon is UUG, although related genes have AUG as the start codon. Changing the start codon to AUG increased expression of cat-86 by 36% in Bacillus subtilis. Changing the start codon to GUG and CUG decreased expression to 65% and 30%, respectively, of the level obtained when AUG was the start codon. CUG has not been previously shown to function as a start codon in B. subtilis. N-terminal sequencing of purified CAT protein specified by the CUG mutant, revealed that CUG was indeed the start codon and specified methionine. The gene xylE, which specifies catechol 2,3-dioxygenase, has AUG as its start codon. Changing the start codon for xylE to CUG decreased expression by 98%. However, when the ribosome-binding site sequence for xylE was optimized and the spacing between it and the start codon was increased to 8 nucleotides, xylE activity increased to 13% of the activity observed for AUG. CUG did not function efficiently as a start codon for cat-86 in Escherichia coli. These data suggest conditions under which CUG can function, with modest efficiency, as a start codon in B. subtilis.

Bacillus subtilis↗

PubMed2004

AUG codons at the beginning of protein coding sequences are frequent in eukaryotic mRNAs with a suboptimal start codon context.

MOTIVATION: The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. However, mRNAs with a suboptimal context of start AUG codon are relatively abundant. It is likely that at least some mRNAs with suboptimal start codon context contain the other signals providing additional information for efficient AUG recognition. RESULTS: Frequency of AUG codons at the beginning of the coding part of eukaryotic mRNAs was analyzed in relation to the context of translation start codon. It was found that the observed downstream AUG content in the mRNAs with optimal start codon context was close to the expected value, whereas it was significantly higher in the mRNAs with a suboptimal context. It is likely that downstream AUG codons can often be utilized as additional start sites to increase translation rate of mRNAs with a suboptimal context of the annotated start codon and many eukaryotic proteins can be characterized by some N-end heterogeneity.

Adenine↗

PubMed2001

Presence of ATG triplets in 5' untranslated regions of eukaryotic cDNAs correlates with a 'weak' context of the start codon.

MOTIVATION: The context of the start codon (typically, AUG) and the features of the 5' Untranslated Regions (5' UTRs) are important for understanding translation regulation in eukaryotic mRNAs and for accurate prediction of the coding region in genomic and cDNA sequences. The presence of AUG triplets in 5' UTRs (upstream AUGs) might effect the initiation rate and, in the context of gene prediction, could reduce the accuracy of the identification of the authentic start. To reveal potential connections between the presence of upstream AUGs and other features of 5' UTRs, such as their length and the start codon context, we undertook a systematic analysis of the available eukaryotic 5' UTR sequences. RESULTS: We show that a large fraction of 5' UTRs in the available cDNA sequences, 15-53% depending on the organism, contain upstream ATGs. A negative correlation was observed between the information content of the translation start signal and the length of the 5' UTR. Similarly, a negative correlation exists between the 'strength' of the start context and the number of upstream ATGs. Typically, cDNAs containing long 5' UTRs with multiple upstream ATGs have a 'weak' start context, and in contrast, cDNAs containing short 5' UTRs without ATGs have 'strong' starts. These counter-intuitive results may be interpreted in terms of upstream AUGs having an important role in the regulation of translation efficiency by ensuring low basal translation level via double negative control and creating the potential for additional regulatory mechanisms. One of such mechanisms, supported by experimental studies of some mRNAs, includes removal of the AUG-containing portion of the 5' UTR by alternative splicing. AVAILABILITY: An ATG_ EVALUATOR program is available upon request or at www.itba.mi.cnr.it/webgene. CONTACT: rogozin@ncbi.nlm.nih.gov, milanesi@itba.mi.cnr.it.

5' Untranslated Regions↗

PubMed2000

Analysis of oligonucleotide AUG start codon context in eukariotic mRNAs.

The AUG start codon context features have been investigated by analyzing eukaryotic mRNAs belonging to various taxonomic groups. The functional relevance of each specific position surrounding the AUG start codon has been established as a function of the measured shift between base composition observed at that particular position, and base composition averaged over all the 5'untranslated regions. A more detailed analysis carried out on human genes belonging to different isochores showed significant isochore-specific fea-tures that cannot be explained only by a mutational bias effect. The most represented heptamers spanning from position -3 to +4 with respect to the initiator AUG have been determined for mRNAs belonging to different taxonomic groups and a web page utility has been set up (http://bigarea.area.ba.cnr.it:8000/BioWWW/ATG.html) to determine the relative abundance of a user submitted oligonucleotide context in a given species or taxon.

5' Untranslated Regions↗

PubMed1999

On negative selection against ATG triplets near start codons in eukaryotic and prokaryotic genomes.

The frequencies of ATG triplets in the genomes of various species were systematically analyzed, and the frequency of ATG triplets was significantly low around start codons in both prokaryotic and eukaryotic genomes. In eukaryotes, however, the frequency decrease before the start codon is much more evident than that after the start codon. In prokaryotes, on the other hand, the ATG frequency pattern around the start codon is less evident, and-more importantly-symmetric. We also computed average distances between a start codon and its nearest upstream-located ATG triplet and found a general tendency for the average distances to be longer in higher organisms.

Animals↗

PubMed2024

Mapping Start Codons of Small Open Reading Frames by N-Terminomics Approach.

sORF-encoded peptides (SEPs) refer to proteins encoded by small open reading frames (sORFs) with a length of less than 100 amino acids, which play an important role in various life activities. Analysis of known SEPs showed that using non-canonical initiation codons of SEPs was more common. However, the current analysis of SEP sequences mainly relies on bioinformatics prediction, and most of them use AUG as the start site, which may not be completely correct for SEPs. Chemical labeling was used to systematically analyze the N-terminal sequences of SEPs to accurately define the start sites of SEPs. By comparison, we found that dimethylation and guanidinylation are more efficient than acetylation. The ACN precipitation and heating precipitation performed better in SEP enrichment. As an N-terminal peptide enrichment material, Hexadhexaldehyde was superior to CNBr-activated agarose and NHS-activated agarose. Combining these methods, we identified 128 SEPs with 131 N-terminal sequences. Among them, two-thirds are novel N-terminal sequences, and most of them start from the 11-31st amino acids of the original sequence. Partial novel N-termini were produced by proteolysis or signal peptide removal. Some SEPs' transcription start sites were corrected to be non-AUG start codons. One novel start codon was validated using GFP-tag vectors. These results demonstrated that the chemical labeling approaches would be beneficial for identifying the start codons of sORFs and the real N-terminal of their encoded peptides, which helps better understand the characterization of SEPs.

Open Reading Frames↗

PubMed1992

Conserved sequence blocks 5' to start codons of plant mitochondrial genes.

Three sequence blocks of 10-12 bp are conserved in sequence and order 5' to putative start codons of several higher-plant mitochondrial genes. At least 25 examples were found, primarily associated with coxII, atp6, and orf25, in monocotyledons and dicotyledons. The proximal block can be 9 bp from start codons, and the three blocks generally occur within 100 bp 5' of start codons. In three examples 5' termini of the blocks represent recombination breakpoints, resulting in conservation of the blocks in resultant configurations. The two proximal blocks can form a secondary structure motif. The occurrence of the blocks near start codons, and conserved sequence and order, is consistent with a possible role in translation initiation or regulation.

Base Sequence↗

PubMed2004

A stable upstream stem-loop structure enhances selection of the first 5'-ORF-AUG as a main start codon for translation initiation of human ACAT1 mRNA.

Human ACAT 1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG(1397-1399) and AUG(1415-1417), at 5'-terminus of the open reading frame (ORF, nt 1397-3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent inframe AUGs at 5'-terminus of the predicted ORF (5'-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5'-ORF-AUGs can be selected as start codons but the first 5'-ORF-AUG(1397-1399) is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5'-ORF-AUG(1397 -1399) as a main start codon, in addition to upstream nucleotide A in the -3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5'-ORF-AUGs. This work showed that a stable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5'-ORF-AUGs.

5' Untranslated Regions↗

PubMed2005

A conformational change in the eukaryotic translation preinitiation complex and release of eIF1 signal recognition of the start codon.

During eukaryotic translation initiation, ribosomal 43S complexes scan mRNAs for the correct AUG codon at which to begin translation. Start codon recognition triggers GTP hydrolysis, committing the complex to engagement at that point on the mRNA. While fidelity at this step is essential, the nature of the codon recognition event and the mechanism by which it activates GTP hydrolysis are poorly understood. Here we report the changes that occur within the 43S.mRNA complex in response to AUG codon recognition. eIF1 and eIF1A are key players in assembly of 43S.mRNA complexes capable of locating initiation codons. We observed FRET between these two factors when bound to the 40S subunit. Using steady-state FRET, anisotropy, and kinetic analyses, we demonstrate that start codon recognition results in a conformational change and release of eIF1 from the ribosome. These rearrangements probably play a role in triggering GTP hydrolysis and committing the complex to downstream events.

Codon, Initiator↗

PubMed1998

Discrimination of 5'-terminal start codons by translation initiation factor 3 is mediated by ribosomal protein S1.

The interrelation between ribosomal protein S1 and IF3 in recognition/discrimination of 5'-terminal start codons by 30S ribosomes has been studied using in vitro toeprinting. The study has been performed with two naturally occurring leaderless mRNAs, lambda cI and phage r1t rro mRNA, as well as with an artificial leaderless mRNA derived from the E. coli ompA gene. We show that in the absence of S1, IF3 does not discriminate against the authentic 5'-terminal start codon of both cI and rro mRNA. Since IF3 was able to exert its proofreading function for initiator tRNA(fMet) on 30S ribosomes lacking S1, this observation cannot be attributed to a lack of binding to or action of IF3 on 30S(-S1) ribosomes. In contrast to leaderless mRNAs, ternary complex formation occurs in the presence of IF3 with 30S ribosomes when the start codon is preceded by a short 20-nucleotide 5'-untranslated region containing a canonical Shine and Dalgarno sequence. This suggests that 5'-terminal start codons are recognised by IF3 as non-standard because of the lack of 16S rRNA-mRNA contacts.

Bacteriophage lambda↗

PubMed2005

Analysis of 5' UTR composition and gene expression: canonical versus non-canonical start codons.

The overall composition upstream of start codons in Escherichia coli was evaluated and viewed in connection with global transcriptome data. Genes starting with AUG as initiation codon tended to be expressed at higher levels than the non-AUG genes, and the upstream region of the non-AUG genes showed negligible signs of Shine-Dalgarno sequences. The latter is in sharp contrast to the AUG genes. Viewing these findings in connection with the current literature, it is proposed that a distinct mechanism for initiation of translation might exist for non-AUG genes that are not preceded by a Shine-Dalgarno sequence. A survey covering a range of other eubacteria (Firmicutes, Proteoacteria, and Actinobacteria) reveals that it is mainly among the Proteobacteria that non-AUG genes do not display clear signs of Shine-Dalgarno regions.

5' Untranslated Regions↗

PubMed2003

The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli.

Antisense peptide nucleic acids (PNA) can inhibit bacterial gene expression with gene and sequence specificity. Using attached carrier peptides that aid cell permeation, the antisense effects when targeting essential genes are sufficient to prevent growth and even kill bacteria. However, many design uncertainties remain, including the difficult question of target sequence selection. In this study, we synthesized 90 antisense peptide-PNAs to target sequences in a head to tail manner across the entire length of the mRNA encoding beta-lactamase. The results from this scan pointed to the start codon region as most sensitive to inhibition. To confirm and refine the result, a higher-resolution scan was conducted over the start codon region of the beta-lactamase gene and the essential Escherichia coli acpP gene. For both genes, the start codon region, including the Shine-Dalgarno motif, was sensitive, whereas antisense agents targeted outside of this region were largely ineffective. These results are in accord with natural antisense mechanisms, which typically hinder the start codon region, and the sensitivity of this region should hold true for most bacterial genes as well as for other RNase H-independent antisense agents that rely on a steric blocking mechanism. Therefore, although other design parameters are also important, the start codon region in E. coli mRNA is the most reliable target site for antisense PNAs.

Acyl Carrier Protein↗

PubMed2003

[Association of the vitamin D receptor gene start codon polymorphism with vitamin D deficiency rickets].

OBJECTIVE: Vitamin D deficiency rickets often causes growth retardation, impaired bone formation and hypocalcemia in children. It is well known that rickets is mainly caused by vitamin D deficiency, but whether there is hereditary susceptibility of children to develop vitamin D deficiency rickets is unknown. Vitamin D receptor (VDR) gene has been used as one of genetic markers in studying the metabolic diseases of bone. The present study aimed to explore the hereditary susceptibility of children to develop rickets through studying the association between VDR gene start codon polymorphism and vitamin D deficiency rickets, METHODS: The subjects were selected from Kunming city, every subject was of Han ethnic group. The subjects were composed of two groups, the patient group consisted of 48 children with active vitamin D deficiency rickets which was diagnosed clinically and confirmed radiologically; the control group was composed of 92 normal children. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), DNA sequence analysis and genetic analysis methods were used. A restriction fragment length polymorphism in the start codon of VDR gene (FokI) was tested in both groups. RESULTS: VDR gene start codon polymorphism was tested successfully for every subject. Frequencies of FF, Ff and ff genotypes were 46%, 33% and 21% in the rickets group, and 22%, 52% and 26% in the control group, respectively. A significant difference was found in the frequency distribution of VDR genotype between two groups (chi(2) = 8.912, P = 0.012). In the patient group, Ff and ff genotypes were less common than control group, but the FF genotype was more common than control group (OR = 3.046), indicating that FF genotype may be significantly associated with vitamin D deficiency rickets. Moreover, VDR allele frequencies of FokI polymorphism also showed significant difference between the two groups (chi(2) = 5.451, P = 0.020), F alleles were more common in patient group than in control group. DNA sequence analysis identified that the start codon of F allele was mutated from ATG to ACG. CONCLUSION: There is an association between VDR gene start codon polymorphism and vitamin D deficiency rickets. This study suggested the possibility that VDR gene polymorphism might be important in determining an individual's susceptibility to development of vitamin D deficiency rickets.

Base Sequence↗

PubMed2002

No association of vitamin D receptor gene start codon fok 1 polymorphisms in Chinese patients with systemic lupus erythematosus.

OBJECTIVE: To examine whether the start codon Fok I of the vitamin D receptor (VDR) gene polymorphism is a marker of susceptibility to or severity of systemic lupus erythematosus (SLE) in Chinese patients in Taiwan. METHODS: A control group of 90 healthy people and 52 patients with SLE were examined. Using polymerase chain reaction (PCR) based restriction analysis, we evaluated the relationship between Fok I polymorphisms and SLE, where an unexcisable length was determined to be 265 bp (FF), and the 2 fragments measuring 169 bp and 96 bp were determined to be excisable lengths (ff). RESULTS: For the genotype of VDR Fok I start codon polymorphism, there were no statistically significant differences between the 2 groups (chi-squared test, p = 0.07). Additionally, we did not detect any association of VDR genotype with the clinical and laboratory profiles in SLE patients. CONCLUSION: Our results suggest that the vitamin D receptor Fok I start codon polymorphism is not related to patients with SLE in Taiwan.

Asian People↗

PubMed1999

Involvement of the aphthovirus RNA region located between the two functional AUGs in start codon selection.

Initiation of translation in picornavirus RNAs occurs internally, mediated by an element termed internal ribosome entry site (IRES). In the aphthovirus RNA, the IRES element directs translation initiation at two in-frame AUGs separated by 84 nucleotides. We have found that bicistronic constructs that contained the IRES element followed by the fragment including the aphthovirus start codons in front of the second gene mimicked the translation initiation pattern of viral RNA observed in infected cells. In those constructs, the frequency of initiation at the first AUG was increased by a sequence context that resembled the favorable consensus for cap-dependent translation, although initiation at the second site was always preferred. In addition, we have found that initiation at the second start codon was not diminished under conditions in which the first initiation codon was blocked by antisense oligonucleotide interference. Interestingly, mutations that positioned the second AUG out-of-frame with the first AUG did not interfere with the frequency of initiation at the second one. On the contrary, IRES-dependent translation initiation in bicistronic constructs lacking the sequences present between functional AUGs in the viral RNA was sensitive to the presence of out-of-frame initiator codons and hairpins in the spacer region. This remarkable difference in start codon recognition was due to the nucleotide composition of the RNA that separated the IRES from the initiator codon. Thus our results indicate that the region located in the aphthovirus RNA between functional AUGs is involved in start codon recognition, strongly favoring selection of the second start AUG as the main initiator codon.

Animals↗

PubMed1990

Changing the start codon context of the 30K gene of tobacco mosaic virus from "weak" to "strong" does not increase expression.

The translation initiation region of the 30K gene of tobacco mosaic virus (TMV) was modified by in vitro mutagenesis to create more optimal start codon contexts. A complicating factor was that modifications in this region also altered the 3' terminus of the 183K ORF that overlaps the 30K ORF. An insertion of GACUCGA between nucleotides 4901 and 4902 resulted in a purine (G) in position -3 relative to the AUG creating a "stronger" start codon context, but this also changed the last four amino acids of the 183K protein. This mutant was infectious, replicated efficiently, but produced reduced amounts of 30K protein. Despite the reduced amount of movement protein, this mutant spread effectively from cell to cell and had a phenotype indistinguishable from that of wild-type virus. A more conservative mutation inserted GAC between TMV nucleotides 4901 and 4902 resulting in a "strong" start codon context (ACGAUGG) and modification of the 183K protein only by insertion of an aspartic acid adjacent to a native aspartic acid. This modification did not enhance the production of 30K protein. These data demonstrate consensus sequences that are optimal for other eukaryotic systems did not cause increased expression of the 30K gene in vivo. The modified sequences of both mutants were stably maintained during relatively long periods of replication. Even though each mutant replicated efficiently, when mixed with wild-type TMV, neither mutant effectively competed with the wild-type virus. Another mutant which removed the native 30K AUG to determine whether subsequent internal start codons with "stronger" contexts would function in its absence was constructed. However, this mutant and a mutant that fused the 183K reading frame to the 30K reading frame did not replicate and move in intact plants.

Base Sequence↗

PubMed2006

An overabundance of phase 0 introns immediately after the start codon in eukaryotic genes.

BACKGROUND: A knowledge of the positions of introns in eukaryotic genes is important for understanding the evolution of introns. Despite this, there has been relatively little focus on the distribution of intron positions in genes. RESULTS: In proteins with signal peptides, there is an overabundance of phase 1 introns around the region of the signal peptide cleavage site. This has been described before. But in proteins without signal peptides, a novel phenomenon is observed: There is a sharp peak of phase 0 intron positions immediately following the start codon, i.e. between codons 1 and 2. This effect is seen in a wide range of eukaryotes: Vertebrates, arthropods, fungi, and flowering plants. Proteins carrying this start codon intron are found to comprise a special class of relatively short, lysine-rich and conserved proteins with an overrepresentation of ribosomal proteins. In addition, there is a peak of phase 0 introns at position 5 in Drosophila genes with signal peptides, predominantly representing cuticle proteins. CONCLUSION: There is an overabundance of phase 0 introns immediately after the start codon in eukaryotic genes, which has been described before only for human ribosomal proteins. We give a detailed description of these start codon introns and the proteins that contain them.

Animals↗

PubMed2006

RNA secondary structure in the coding region of dengue virus type 2 directs translation start codon selection and is required for viral replication.

Dengue virus is a positive-strand RNA virus and a member of the genus Flavivirus, which includes West Nile, yellow fever, and tick-borne encephalitis viruses. Flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (C) protein. Dengue virus type 2 (DENV2) and other mosquito-borne flaviviruses initiate translation of C from a start codon in a suboptimal context and have multiple in-frame AUGs downstream. Here, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region cis element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.

Animals↗