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PubMed2006

Naturally occurring adenines within mRNA coding sequences affect ribosome binding and expression in Escherichia coli.

Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many Escherichia coli genes, and addition of downstream adenine-rich sequences increases expression from several genes in E. coli. Here we describe site-directed mutagenesis of the E. coli aroL, pncB, and cysJ coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from aroL-, pncB-, and cysJ-lacZ fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of E. coli translation.

Adenine↗

PubMed1996

Construction of the complete rat fatty acid synthase cDNA and its expression in Saccharomyces cerevisiae.

The 272 647-dalton polypeptide of fatty acid synthase (FAS) from Rattus norvegicus has been expressed in a proteinase-deficient strain of Saccharomyces cerevisiae. The seven overlapping cDNA clones for rat FAS spanning the entire coding region were the starting material for this undertaking. In a series of cloning steps an expression plasmid was constructed in which the cDNA was placed under the control of the yeast ADH1 promoter. Northern blotting of total RNA isolated from yeast transformed with this expression plasmid demonstrated a high rate of transcription of the 7.4-kb cDNA. However, a successful translation required further manipulation of the sequence immediately upstream of the rat FAS translational start codon. This was obtained when the 86 bp of the rat FAS cDNA immediately 5' to the start codon were replaced by a nonamer corresponding to the immediate 5'-vicinity of the translational start codon of the yeast ADH1 gene. Nevertheless, the translation product could be detected only by Western blotting. The FAS proteins of S. cerevisiae and rat are not functionally interchangeable. Using the purification protocol of rat FAS the heterologously expressed FAS could be enriched by at least one order of magnitude.

Animals↗

PubMed1996

Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5' untranslated leader.

The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.

Amino Acid Sequence↗

PubMed2001

Construction of a multiframe vector to express coding sequences in Escherichia coli.

Cloning of foreign DNA fragments for coding sequence analysis in Escherichia coli usually involves sets of three vectors. To simplify this, we constructed an expression vector named pMFV7 containing three ATG codons in different frames downstream of a Shine-Dalgarno sequence, assuming that the ribosome can use any of the three start codons in an alternative manner. Translation beginning at either of the start codons would drive the expression of any coding fragment cloned downstream. To test the feasibility of this proposal, we cloned DNA fragments of the lacZ gene in each of the possible reading frames downstream from pMFV7 start codons. Sequence analysis of the N-terminus regions around the fusion sites indicates that ribosomes indeed initiate translation at each of the three initiation codons. In one case, levels of beta-galactosidase activity depended largely on the N-terminus of the translation products. We conclude that pMFV7 may be useful for expressing coding sequences regardless of their reading frame.

Cloning, Molecular↗

PubMed2004

Multiple elements required for translation of plastid atpB mRNA lacking the Shine-Dalgarno sequence.

The mechanism of translational initiation differs between prokaryotes and eukaryotes. Prokaryotic mRNAs generally contain within their 5'-untranslated region (5'-UTR) a Shine-Dalgarno (SD) sequence that serves as a ribosome-binding site. Chloroplasts possess prokaryotic-like translation machinery, and many chloroplast mRNAs have an SD-like sequence, but its position is variable. Tobacco chloroplast atpB mRNAs contain no SD-like sequence and are U-rich in the 5'-UTR (-20 to -1 with respect to the start codon). In vitro translation assays with mutated mRNAs revealed that an unstructured sequence encompassing the start codon, the AUG codon and its context are required for translation. UV crosslinking experiments showed that a 50 kDa protein (p50) binds to the 5'-UTR. Insertion of an additional initiation region (SD-sequence and AUG) in the 5'-UTR, but not downstream, arrested translation from the authentic site; however, no inhibition was observed by inserting only an AUG triplet. We hypothesize for translational initiation of the atpB mRNA that the ribosome enters an upstream region, slides to the start codon and forms an initiation complex with p50 and other components.

5' Untranslated Regions↗

PubMed2002

Correlations between Shine-Dalgarno sequences and gene features such as predicted expression levels and operon structures.

This work assesses relationships for 30 complete prokaryotic genomes between the presence of the Shine-Dalgarno (SD) sequence and other gene features, including expression levels, type of start codon, and distance between successive genes. A significant positive correlation of the presence of an SD sequence and the predicted expression level of a gene based on codon usage biases was ascertained, such that predicted highly expressed genes are more likely to possess a strong SD sequence than average genes. Genes with AUG start codons are more likely than genes with other start codons, GUG or UUG, to possess an SD sequence. Genes in close proximity to upstream genes on the same coding strand in most genomes are significantly higher in SD presence. In light of these results, we discuss the role of the SD sequence in translation initiation and its relationship with predicted gene expression levels and with operon structure in both bacterial and archaeal genomes.

Archaeal Proteins↗

PubMed2004

The fertility restorer genes X and T alter the transcripts of a novel mitochondrial gene implicated in CMS1 in chives (Allium schoenoprasum L.).

A chimeric mitochondrial gene configuration, mainly derived from sequences associated with the essential genes atp9 and atp6, was isolated from the sterility-inducing cytoplasm of the CMS1 system in chives (Allium schoenoprasum L.). This sequence is not found in four other cytoplasm types from chives; however, two copies are present in the mitochondrial DNA of CMS1-inducing cytoplasm, whose 5'-sequences are homologous to those of the atp9 gene. We provide evidence to show that one of the two CMS1-specific copies is actively transcribed, and two transcripts which terminate at the same position but differ in their 5'initiation sites were localized using the RACE technique. These transcripts of 942 and 961 nt, respectively, were confirmed to be the major products of this gene in CMS1 plants by Northern hybridization. However, smaller transcripts were found to accumulate in plants in which fertility had been restored. Restoration of fertility was induced either by the gene X, or the gene T at high temperatures. In (S1) X. genotypes a transcript with an estimated size of 440 nt was detected in all tissues examined. An additional hybridization signal with an estimated size of approximately 850 nt is expressed in temperature-sensitive plants [(S1) xxT.], and the intensity of a minor 350-nt transcript is enhanced. These latter alterations, conditioned by the gene T, occur independently of the growth temperature, but are limited to the flowers; they were not observed in leaves. The CMS1 transcripts are edited at seven positions and contain an ORF with a maximum coding capacity of 780 nt (containing the start codon derived from the atp9 gene in-frame). Use of the third in-frame start codon would result in the synthesis of a protein of a size very close to that of a previously described CMS1-specific protein, which has an apparent molecular weight of 18 kDa. The coding sequence that begins at this third in-frame start codon is also present in the sterility-inducing cytoplasms (S) and (T) in the onion, and absent in (N) cytoplasm.

Amino Acid Sequence↗

PubMed1999

Genomic location and nucleotide sequence of a serotype 3 porcine adenovirus hexon gene.

The putative hexon gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and the nucleotide sequence determined. The genomic location of the PAV3 hexon gene was determined and an open reading frame (ORF) encoding a polypeptide of 939 amino acids identified. Comparison of the nucleotide sequence of the putative PAV3 hexon gene with the sequence of the HAV2 hexon gene returned an overall identity of approximately 63%. A stop codon 144 nucleotides upstream and a start codon 18 nucleotides downstream of the ORF were identified and comparison with the HAV genome demonstrated that their positions corresponded to the stop site of the pVI gene and start site of the 23K gene, respectively. To confirm the correct start codon of the putative PAV3 hexon gene, the acceptor splice site for the putative PAV3 hexon gene was determined from cDNA and found to be between the two guanines immediately upstream of the first ATG in the ORF. Comparison with the HAV2 hexon protein showed overall identity of approximately 65%, with higher identity in the carboxy-terminus of approaching 76% over 380 amino acids. Multiple alignment of the PAV3 hexon amino acid sequence with other known HAV and animal adenovirus hexon sequences indicated that conservation is generally maintained but that identity is much lower within the loop structures of the protein.

Amino Acid Sequence↗

PubMed1994

Analysis of the role of the Shine-Dalgarno sequence and mRNA secondary structure on the efficiency of translational initiation in the Euglena gracilis chloroplast atpH mRNA.

Chloroplast mRNAs in Euglena gracilis fall into two classes. One class has a Shine-Dalgarno sequence 5' to the AUG start codon while the other group of mRNAs does not have any conserved sequence elements near the start codon. The chloroplast mRNA encoding the atpH gene has been selected as an example of a message which has a Shine-Dalgarno sequence (GGAGUU) located in the initiation region. Mutations in the Shine-Dalgarno sequence result in 2-5-fold reductions in the efficiency of the message in initiation complex formation depending on the precise mutation. Secondary structure mapping of the initiation region of the atpH mRNA suggests that a number of mRNA conformations are present in an equilibrium mixture. Mutations in the Shine-Dalgarno sequence had little effect on the overall structure of the mRNA. Directed mutations which place the Shine-Dalgarno sequence into a region of higher secondary structure drastically reduce the activity of the message. The monocistronic form of the atpH mRNA has a 5'-untranslated leader region slightly over 70 nucleotides in length. Deletions in the leader suggest that a minimum length of 10 bases 5' to the start codon is important for activity in initiation. The presence of the full leader increased efficiency in initiation about 2-fold compared to messages with leaders 25-40 residues in length.

Animals↗

PubMed1998

Small genes/gene-products in Escherichia coli K-12.

Forty-two protein spots of observed M(r) 6-15 kDa were resolved by two-dimensional gel electrophoresis, stained by Coomassie blue and subjected to Edman microsequencing. All of the proteins could be related back to their encoding open reading frames, thereby vindicating the bioinformatic tools currently utilised in their identification. However, only 14/42 gene-products were expressed as annotated. Translation was confirmed for 14 open reading frames with no attributed function (EcoGene Y-entries), while N-terminal sequence allowed the start codon to be accurately annotated for the genes yigF, yccU, yqiC, ynfD, and yeeX. The methionine start codon was cleaved in 11 gene-products (AtpE, Hns, RpoZ, RplL, CspC, YccJ, YggX, YjgF, HimA, InfA, RpsQ) and a further five showed loss of a signal peptide (PspE, HdeB, HdeA, YnfD, YkfE). Internal (Tig, AtpA, TufA) and N-terminal fragmentation (CspD, RpsF, AtcU) of much larger proteins was also detected, which may have resulted from physiological or translational processes. M(r) and pI isoforms were detected respectively for PtsH and GatB, each being phosphoproteins, as well as RplY which manifested differences with respect to predicted M(r) and pI. In addition, YjgF was shown to belong to a small gene family of unknown function with ancient conserved regions across procaryotes and eucaryotes. YgiN was revealed to have a paralogue and orthologues in Bacillus subtilis, Synechocystis sp., Mycobacterium tuberculosis, Neisseria gonorrhoea, and Rhodococcus erythropolis. Orthologues are also reported for YihD, YccU and YeeX. Of the 14 Y-genes, only YkfE possessed no detectable orthologues. These results highlight the need to complement genomic analysis with detailed proteomics in order to gain a better understanding of cellular molecular biology, while the confirmation of the open reading frame start codon using Edman degradation protein microsequencing has yet to be superseded by recent advances in mass spectrometry.

Amino Acid Sequence↗

PubMed2006

The short vitamin D receptor is associated with increased risk for generalized aggressive periodontitis.

BACKGROUND: Generalized aggressive periodontitis (GAP) exhibits severe inflammation and alveolar bone loss. Vitamin D receptor (VDR) regulates both bone metabolism and inflammation-related genes, and its polymorphisms and haplotypes may affect the functional activity of the VDR protein in GAP. OBJECTIVE: We analysed the genetic effect of VDR start codon, intron, and exon polymorphisms, and their haplotypes on the development of GAP. MATERIALS AND METHODS: The VDR start codon 27823C > T (rs2228570, FokI), intron 8 60890G > A (rs154410, BsmI), and exon 9 61968T > C (rs731236, TaqI) polymorphisms were determined by using the polymerase chain reaction-restriction fragment length polymorphism analysis among 93 GAP patients and 143 healthy controls. RESULTS: The VDR start codon 27823*C/*C genotype was associated with an increased risk for GAP [odds ratio (OR) = 1.83, p = 0.028], but the intron 8 60880G > A and exon 9 61968T > C polymorphisms were not associated with GAP. The VDR haplotype homozygote ht1(C-G-T) carrying 27823*C allele was associated with a 1.8-fold increased risk of GAP (OR = 1.84, p = 0.030). CONCLUSION: These results demonstrate that the short VDR (27823*C/*C) protein may influence GAP susceptibility.

Adenine↗

PubMed2004

Transcriptional and translational analysis of the ccaR gene from Streptomyces clavuligerus.

CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in Streptomyces clavuligerus. Previous sequence analyses of the ccaR gene revealed two possible start codons, an ATG, and a GTG located in-frame 18 bp downstream of the ATG. To determine the true start codon, ccaR was expressed, either from the GTG or ATG codon, in Escherichia coli. A protein product was only obtained from the ATG construct. Similarly, ccaR constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in Streptomyces species were prepared and used to complement a S. clavuligerus ccaR mutant. Bioassays showed that only the ATG construct could complement the ccaR mutant to restore cephamycin C production, and Western analysis confirmed the presence of CcaR in the mutant complemented with the ATG construct only. To ensure that expression of ccaR from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into ccaR, converting the GTG to GTC. The GTC construct still fully complemented a ccaR mutant, confirming that ATG is the true start codon. Inspection of the region upstream of ccaR by S1 nuclease protection and primer extension analyses indicated the presence of two transcript start points that mapped to residues located 74 and 173 bp upstream of the ATG codon.

Bacterial Proteins↗

PubMed1987

Potential secondary structure at translation-initiation sites.

Since translational start codons also occur internally, more-complex features within mRNA must determine initiation. We compare the potential secondary structure of 123 prokaryotic mRNA start regions to that of regions coding for internal methionines. The latter display an unexpectedly-uniform, almost-periodic pattern of pairing potential. In contrast, sequences 5' to start codons have little self-pairing, and do not pair extensively with the proximal coding region. Pairing potential surrounding start codons was found to be less than half of that found near internal AUGs. In groups of random sequences where the distribution of nucleotides at each position, or of trinucleotides at each in-frame codon position, matched the observed natural distribution, there was no periodicity in the pairing potential of the internal sequences. Randomized internal sequences had less pairing: the ratio of pairing intensity between internals and starts was reduced from 2.0 to 1.6 by randomization. We propose that the transition from the relatively-unstructured start domains to the highly-structured internal sequences may be an important determinant of translational start-site recognition.

Base Sequence↗

PubMed2001

Physical evidence for distinct mechanisms of translational control by upstream open reading frames.

The Saccharomyces cerevisiae GCN4 mRNA 5'-leader contains four upstream open reading frames (uORFs) and the CPA1 leader contains a single uORF. To determine how these uORFs control translation, we examined mRNAs containing these leaders in cell-free translation extracts to determine where ribosomes were loaded first and where they were loaded during steady-state translation. Ribosomes predominantly loaded first at GCN4 uORF1. Following its translation, but not the translation of uORF4, they efficiently reinitiated protein synthesis at Gcn4p. Adding purified eIF2 increased reinitiation at uORFs 3 or 4 and reduced reinitiation at Gcn4p. This indicates that eIF2 affects the site of reinitiation following translation of GCN4 uORF1 in vitro. In contrast, for mRNA containing the CPA1 uORF, ribosomes reached the downstream start codon by scanning past the uORF. Addition of arginine caused ribosomes that had synthesized the uORF polypeptide to stall at its termination codon, reducing loading at the downstream start codon, apparently by blocking scanning ribosomes, and not by affecting reinitiation. The GCN4 and CPA1 uORFs thus control translation in fundamentally different ways.

5' Untranslated Regions↗

PubMed1995

Nucleotide sequence and transcriptional analysis of the gp41 gene of Spodoptera frugiperda nuclear polyhedrosis virus.

The Spodoptera frugiperda multiple nucleocapsid nuclear polyhedrosis virus (SfMNPV) gp41 structural protein gene was located in the 1.9 kbp EcoRI-S fragment and sequenced. An open reading frame (ORF) of 999 nucleotides was detected that encoded a protein of 332 amino acids. The gp41 gene transcript was detected after 12 h post-infection (p.i.) and remained detectable at 48 h p.i. Two major mRNAs, about 1.6 and 2.8 kb in length, were determined by Northern blot analysis. Primer extension analysis demonstrated that the gp41 gene promoter region contains three transcription start sites. Two of the gp41 gene transcription start sites were located at -42 and -41 nucleotides from the ATG translation start codon within a consensus late transcription start site (TAAG) and another transcription start site was located at -140 nucleotides from the ATG translation start codon for which no consensus motif has been determined. Comparison of SfMNPV gp41 nucleotide and amino acid sequences with the gp41 genes from Autographa californica, Bombyx mori, and Helicoverpa zea NPVs showed 60% homology of nucleotide sequences and 70% similarity of amino acid sequences.

Amino Acid Sequence↗

PubMed1994

Transcriptional mapping of the varicella-zoster virus regulatory genes encoding open reading frames 4 and 63.

Four of the 68 varicella-zoster virus (VZV) unique open reading frames (ORFs), i.e., ORFs 4, 61, 62, and 63, encode proteins that influence viral transcription and are considered to be positional homologs of herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins. In order to identify the elements that regulate transcription of VZV ORFs 4 and 63, the encoded mRNAs were mapped in detail. For ORF 4, a major 1.8-kb and a minor 3.0-kb polyadenylated [poly(A)+] RNA were identified, whereas ORF 63-specific probes recognized 1.3- and 1.9-kb poly(A)+ RNAs. Probes specific for sequences adjacent to the ORFs and mapping of the RNA 3' ends indicated that the ORF 4 RNAs were 3' coterminal, whereas the RNAs for ORF 63 represented two different termination sites. S1 nuclease mapping and primer extension analyses indicated a single transcription initiation site for ORF 4 at 38 bp upstream of the ORF start codon. For ORF 63, multiple transcriptional start sites at 87 to 95, 151 to 153, and (tentatively) 238 to 243 bp upstream of the ORF start codon were identified. TATA box motifs at good positional locations were found upstream of all mapped transcription initiation sites. However, no sequences resembling the TAATGARAT motif, which confers IE regulation upon HSV-1 IE genes, were found. The finding of the absence of this motif was supported through analyses of the regulatory sequences of ORFs 4 and 63 in transient transfection assays alongside those of ORFs 61 and 62. Sequences representing the promoters for ORFs 4, 61, and 63 were all stimulated by VZV infection but failed to be stimulated by coexpression with the HSV-1 transactivator Vmw65. In contrast, the promoter for ORF 62, which contains TAATGARAT motifs, was activated by VZV infection and coexpression with Vmw65. These results extend the transcriptional knowledge for VZV and suggest that ORFs 4 and 63 contain regulatory signals different from those of the ORF 62 and HSV-1 IE genes.

Base Sequence↗

PubMedDate not supplied

Modification in framework region I results in a decreased affinity of chimeric anti-TAG72 antibody.

A mouse/human chimeric B72.3P-1-6 antibody was produced by construction of a novel expression vector mpSV2neo-EP2-V-Crl containing the same gene fragments as the expression vector mpSV2neo-EP1-V-Crl (Xiang J., Roder J. and Hozunni N., submitted to Molec. Immun., 1991) except the promoter (P2) fragment in which the translation start codon ATG is retained. The expression vector was transfected into a heavy chain loss mutant cell line, B72.3M1. The translation of the chimeric heavy chain may start at the exogenous start codon ATG within the P2 fragment, which is 27 base pairs upstream of the endogenous start codon ATG in B72.3 heavy chain V region cDNA fragment, leading to an alteration in leader sequence cleavage sites and the formation of chimeric heavy chain with an elongation in the FR1 region. Chimeric B72.3P-1-6 antibody retained binding specificity to TAG72 antigen, but showed an eight-fold decrease in binding affinity to TAG72 compared with chimeric B72.3-1-3 antibody. This suggests that residues in FRI contribute to the correct folding of the antibody binding region of the B72.3 antibody.

Amino Acid Sequence↗

PubMed1989

Analysis of the promoter region of the melanin locus from Streptomyces antibioticus.

Several approaches were used to study the transcriptional control region of the melanin-production locus (melC) of Streptomyces antibioticus. Filter-binding in combination with exonuclease III protection localized the 3' boundary of a Streptomyces RNA polymerase-binding site predominantly about 39 nucleotides (nt) upstream from the start codon of melC1, the first open reading frame in the melC locus. Deletion of nt 112-197 upstream from the melC1 start codon reduced melC expression to less than 10%, and deletion of nt 28-107 or 28-120 upstream from melC1 totally inactivated melC. High-resolution nuclease S1 mapping identified the in vitro transcriptional start point (tsp) at 33-34 nt upstream from the start codon of melC1. No sequence resembling the E. coli consensus promoter sequence was found in this region, and site-directed mutagenesis of such a sequence located 101-132 nt upstream from melC1 did not influence melC expression. These studies suggest that transcription of melC is principally from a single tsp and is positively regulated by a mechanism that involves sequences 87-163 nt upstream from the tsp.

Base Sequence↗