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PubMed1987

Isolation and characterization of a vinculin cDNA from chick-embryo fibroblasts.

A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3' sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.

Amino Acid Sequence↗

PubMed1997

Cloning of a polycistronic cDNA from tomato encoding gamma-glutamyl kinase and gamma-glutamyl phosphate reductase.

We isolated from a tomato cDNA library the tomPRO1 locus, which encodes gamma-glutamyl kinase (GK) and gamma-glutamyl phosphate reductase (GPR). This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons. The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR. DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1 sequence in tomato. We demonstrated with RNase protection assays that the tomPRO1 locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon. In Escherichia coli, tomPRO1 directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified from E. coli recognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots. These results suggest that in tomato tissues, GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping. The tomPRO1 locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames.

Aldehyde Oxidoreductases↗

PubMed1991

Antibacterial activity and inhibition of protein synthesis in Escherichia coli by antisense DNA analogs.

Protein synthesis, which takes place within ribosomes, is essential for the survival of any living organism. Ribosomes are composed of both proteins and RNA. Specific interaction between the 3' end CCUCC sequence of prokaryotic 16S rRNA and a partially complementary sequence preceding the initiating codon of mRNA is believed to be a prerequisite for initiation of protein synthesis. Here we report the use of short (three to six nucleotides) synthetic DNA analogs complementary to this sequence to block protein synthesis in vitro and in vivo in Escherichia coli. In the DNA analogs the normal phosphodiester bond in the antisense DNA was replaced by methylcarbamate internucleoside linkages to enhance transport across plasma membranes. Of the analogs tested, those with the sequence AGG and GGA inhibit protein synthesis and colony formation by E. coli strains lacking an outer cell wall. Polyethylene glycol 1000 (PEG 1000) was attached to the 5' end of some of the test methylcarbamate DNAs to enhance solubility. Analogs of AGG and GGAG with PEG 1000 attached inhibited colony formation in normal E. coli. These analogs may be useful food additives to control bacterial spoilage and biomedically as antibiotics.

3T3 Cells↗

PubMed2003

High-level expression of porcine liver cytochrome P-450 reductase catalytic domain in Escherichia coli by modulating the predicted local secondary structure of mRNA.

A direct expression system for the solubilized catalytic domains of NADPH-cytochrome P-450 reductase (sCPR) from rat (RsCPR) and porcine (PsCPR) in Escherichia coli cells was constructed using the expression plasmid pCWori(+). PsCPR was minimally expressed, whereas RsCPR was highly expressed. Replacement of the nucleotides encoding Thr(60)Ser(61)Ser(62) in PsCPR with those for Ala(60)Pro(61)Pro(62) in RsCPR markedly increased the expression level of the protein. The local secondary structures of the mRNAs, which were predicted with the prediction program GeneBee (http://www.genebee.msu.su), suggested that the intramolecular double strand between the ribosome binding site (RBS) and the Thr(60)Ser(61)Ser(62) codons in PsCPR, and/or the base-pairing at the initiation codon of the mRNAs significantly affected protein expression. Silent mutations were systematically introduced into the codons for Thr(58) and Thr(60)Ser(61) in PsCPR to modulate the local secondary structure of the mRNA. The expression level of the silently mutated PsCPR suggests that the expression level of PsCPR depends on the stability of the local structure at the RBS in the mRNA. A high-level expression system for wild-type PsCPR was constructed by introducing silent mutations at the codons for Thr(60)Ser(61) in PsCPR. The purified PsCPR showed the characteristic absorption spectral changes of sCPR after reduction with NADPH. The yield of purified PsCPR from 1 liter of culture fluid was 45.8 mg. These results substantiate that the introduction of silent mutations in the section of the gene encoding the N-terminal region of the protein based on the predicted local secondary structure of the mRNA at the RBS is a useful approach to control and increase the expression level of heterologous proteins in E. coli cells.

Amino Acid Sequence↗

PubMed1989

Transcriptional mapping and nucleotide sequence of a vaccinia virus gene encoding a polypeptide with extensive homology to DNA ligases.

Nucleotide sequencing of the vaccinia virus SalI F DNA fragment identified an open reading frame of 552 amino acids encoding a protein of 63.3 kDa. The deduced amino acid sequence shares 30% identity with S. pombe and S. cerevisiae DNA ligases, with homology strongest near the carboxy terminus and around the lysine residue required for ligase-adenylate formation. Prokaryotic DNA ligases are poorly related to the vaccinia sequence. The initiation codon of the ORF forms part of a late transcriptional initiation sequence TAAATG and is preceded by two overlapping early transcriptional termination signals, TTTTTTTAT. Nonetheless, RNA mapping showed that the ligase gene is transcribed early during infection and the 5' end of the mRNA maps to the TAAATG motif. The possible roles of a DNA ligase in vaccinia virus DNA replication and recombination are discussed.

Amino Acid Sequence↗

PubMed1989

Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.

Amino Acid Sequence↗

PubMed1990

Sulfate and thiosulfate transport in Escherichia coli K-12: identification of a gene encoding a novel protein involved in thiosulfate binding.

The sequence of 1,973 nucleotides encompassing the region at and directly adjacent to the CysB-dependent promoter controlling expression and synthesis of the sulfate-thiosulfate transport system of Escherichia coli has been determined. The transcription start site has been mapped by primer extension. One open reading frame representing the first gene of the presumed sulfate transport operon was identified and designated cysP. The deduced amino acid sequence of the CysP polypeptide indicates the presence of a signal peptide. Expression of the cysP gene in the T7 promoter-polymerase system revealed the location of the gene product in the periplasm. Construction of a cysP insertional mutant and assays of binding and uptake of sulfate and thiosulfate by this mutant allowed the identification of the cysP gene product as a thiosulfate-binding protein. The TGA termination codon of cysP was found to overlap the putative ATG initiation codon of the next open reading frame, inferred as being essential for the sulfate transport system, and it was designated cysT. Preliminary sequence data from the corresponding region of the Salmonella typhimurium chromosome showed strictly homologous counterparts of the E. coli cysP and cysT genes.

Amino Acid Sequence↗

PubMed1995

Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets.

Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.

Base Sequence↗

PubMed1988

The structural genes coding for the L and M subunits of Rhodospirillum rubrum photoreaction center.

In Rhodospirillum rubrum, pufL, and pufM, the structural genes coding for the photoreaction center L and M polypeptides, are comprised respectively of 831 and 921 nucleotides. They are separated by a stretch of 12 nucleotides between the TAA stop codon of pufL and the first base of the ATG initiation codon of pufM. The predicted amino acid sequence of the L and M polypeptides, respectively, contain 275 and 305 residues with corresponding molecular weights of 30,473 and 33,978. Their sequences are highly homologous to those of Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis. As can be deduced from the crystallographic structure of other photoreaction centers, the regions of greatest similarity are the binding sites of the cofactors involved in the photochemical reaction rather than the protein secondary structure. L and M contain, at conserved positions of their sequences, three main clusters of positively charged residues on the cytoplasmic side of the membrane. This arrangement may be involved in protein orientation during membrane assembly. Evolutionary distance of pufL and pufM, as assessed by substitution frequency analysis, confirms the closeness of the two Rhodobacter species, the other two species being equidistant from one another. Interspecies evolutionary distance is greater for pufL than for pufM.

Bacterial Proteins↗

PubMed1983

mRNA secondary structure as a determinant in cap recognition and initiation complex formation. ATP-Mg2+ independent cross-linking of cap binding proteins to m7I-capped inosine-substituted reovirus mRNA.

Polypeptides of Mr = 50,000 and 80,000 in rabbit reticulocyte initiation factor preparations can be specifically cross-linked to the oxidized t' cap structure of native reovirus mRNA in an ATP-Mg2+-dependent manner (Sonenberg, N., Guertin, D., Cleveland, D., and Trachsel, H. (1981) Cell 27, 563-572). However, specific cross-linking of these polypeptides can occur in the absence of ATP-Mg2+ when m7I-capped inosine substituted mRNA, which contains less secondary structure than native reovirus mRNA, is used. We also found, using wheat germ extract, that inhibition of initiation complex formation by high salt concentrations is directly related to the degree of secondary structure of the mRNA. Binding of ribosomes to bromouridine-substituted reovirus mRNA is severely inhibited at high K+ concentrations, while binding to inosine-substituted mRNA is only slightly inhibited and binding of native reovirus mRNA is inhibited to an intermediate degree. The results are consistent with the hypothesis that cap recognition factors mediate an ATP-dependent melting of secondary structures involving 5' proximal sequences to the initiation codon in order to facilitate binding of ribosomes during translation initiation.

Adenosine Triphosphate↗

PubMed2002

Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control.

It is commonly believed that the translational efficiency of prokaryotic mRNAs is intrinsically determined by both primary and secondary structures of their translational initiation regions. However, for leaderless mRNAs starting with the AUG initiating codon occurring in bacteria, archaea and eukaryotes, there is no evidence for ribosomal recruitment signals downstream of the 5'-terminal AUG that seems to be the only necessary and constant element. Studies in Escherichia coli have brought to light that the ratio of initiation factors IF2 and IF3 plays a decisive role in translation initiation of leaderless mRNA, indicating that the translational efficiency of this mRNA class can be modulated depending on the availability of components of the translational machinery. Recent data suggested that the start codon of bacterial leaderless mRNAs is recognized by a ribosome-IF2-fMet-tRNA complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes, which points to a conceptual similarity in all initiation pathways. In fact, the faithful translation of leaderless mRNAs in heterologous systems shows that the ability to translate leaderless mRNAs is an evolutionarily conserved function of the translational apparatus.

5' Untranslated Regions↗

PubMed1989

Mapping the lacZ ribosome binding site by RNA footprinting.

The ribosome binding site of the Escherichia coli lacZ mRNA has been characterized by using an RNA footprinting technique. Purified E. coli 70S ribosomes and fMet-tRNA were incubated with mRNA, and the complex was treated with RNA-reactive reagents or RNases as probes. The protected sites on the mRNA were then mapped by extending a radioactive primer with reverse transcriptase. Dimethyl sulfate, diethyl pyrocarbonate, and 1,10-phenanthroline-copper ion oxidative complex were used as reagent probes; they detected interaction sites within the ribosome binding site. A region of approximately 35 nucleotides was protected by the ribosome, specifically across the Shine-Dalgarno region, around the fMet initiation codon, and at a region 7-12 nucleotides distal to the fMet codon. In addition, an enhanced reaction occurred between the fMet codon and the distal site. These results imply an internally selective interaction between the ribosome and the mRNA sequence. The enhanced reactivity of a site distal to the initiation site--flanked by the AUG codon and a site previously identified as conserved in a study of initiation sequences--may indicate a region where the mRNA is specifically exposed.

Alkylating Agents↗

PubMed1989

High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons.

A 6.75-kilobase human hepatoma-derived basic fibroblast growth factor (bFGF) cDNA was cloned and sequenced. An amino-terminal sequence generated from a purified hepatoma bFGF was found to correspond to the nucleotide sequence and to begin 8 amino acids upstream from the putative methionine start codon thought to initiate a 154-amino acid bFGF translation product. This sequence suggests that a form of bFGF of at least 163 amino acids exists. The hepatoma cDNA was transcribed in vitro into RNA; in vitro translation of this RNA generated three forms of bFGF with molecular masses of 18, 21, and 22.5 kDa. By use of in vitro mutagenesis, it was found that the 22.5-kDa bFGF and possibly the 21-kDa form were initiated with CUG start codons. The 18-kDa bFGF was initiated with an AUG codon. By transfecting into COS cells human hepatoma bFGF cDNA and a construct from which the AUG initiator was eliminated, it was found that the higher molecular mass forms of bFGF were as biologically active as the 18-kDa form.

Amino Acid Sequence↗

PubMed2001

Expression of penicillin G acylase from the cloned pac gene of Escherichia coli ATCC11105. Effects of pacR and temperature.

The structural gene pac in Eschericia coli ATCC11105 encodes penicillin G acylase (PGA). Within the pac gene, there is a regulatory gene pacR, which is transcribed in the opposite direction. Site-directed mutagenesis was performed at base 1045 of pac by replacing a T with a C. This substitution did not alter the amino-acid sequence of PGA, but changed the translation start codon of pacR from AUG to GUG. The expression of the mutant pacR decreased dramatically and the lacZ transcriptional fusion analysis showed that GUG was an extremely poor initiation codon for pacR. The pacR mutation caused PGA expression to be constitutive rather than inductive in two strains (E. coli A56, DH10B). The pac inducer phenylacetic acid (PAA) gave significant induction of PGA production at a concentration of 0.2% in wild type, but PAA at this concentration inhibited both cell growth and PGA production in the pacR mutated strains. The temperature-dependent expression character of pac is preserved in the pacR translation-initiation mutant and the optimum temperature of PGA production was 22 degrees C in both wild type and mutant. At a higher temperature of 37 degrees C, the PGA precursor polypeptide could not be matured into subunits and formed inclusion bodies, as revealed by western blot analysis. Our investigations confirmed the hypothesis of pacR-mediated PAA induction for PGA expression and clarified the inhibitory effect of high temperature upon the post-translational processing of the PGA precursor polypeptide.

Bacterial Proteins↗

PubMed1994

The large surface protein of duck hepatitis B virus is phosphorylated in the pre-S domain.

The two major envelope proteins (large [L] and small [S]) of duck hepatitis B virus are encoded by the pre-S/S open reading frame. The L protein is initiated from the AUG at position 801 in the pre-S region of the pre-S/S coding sequence, yielding an N-terminal consensus sequence for myristylation. Western immunoblots of the L protein often reveal a doublet at 36 and 35 kDa, with the latter attributed to the use of one of the three internal initiation codons. However, metabolic labelling with [3H]myristic acid results in labelling of both P35 and P36, indicating that both species must be initiated from the same start codon. Using metabolic labelling with 32P and digestion with residue-specific phosphatases, we demonstrate that L protein heterogeneity is due to phosphorylation of threonine and/or serine residues within the pre-S domain. We propose that at least one possible phosphorylation site is located at a novel (S/T)PPL motif which is conserved near the carboxyl end of the pre-S1 domain in all hepadnavirus sequences. Two to three additional (S/T)P motifs are also present in the carboxyl half of the pre-S1 (but not pre-S2 or S) domain of all hepadnaviruses. L protein in serum-derived particles is resistant to phosphatase digestion in the absence of detergents, reflecting an internal disposition of the phosphorylated pre-S domain and suggesting a role for dephosphorylation in the topological shift within L during morphogenesis (P. Ostapchuk, P. Hearing, and D. Ganem, EMBO J. 13:1048-1057, 1994). Furthermore, we observe that the relative amount of the phosphorylated form of L increases with time in the viral growth cycle. These findings imply that phosphorylation-dephosphorylation of the L protein is an important, regulated mechanism necessary for correct virion morphogenesis.

Animals↗

PubMed1992

Cardioviral internal ribosomal entry site is functional in a genetically engineered dicistronic poliovirus.

High mutation rates have driven RNA viruses to shorten their genomes to the minimum possible size. Mammalian (+)-strand RNA viruses and retroviruses have responded by reducing the number of cis-acting regulatory elements, a constraint that has led to the emergence of the polyprotein. Poliovirus is a (+)-stranded picornavirus whose polyprotein, encoded by an open reading frame spanning most of the viral RNA, is processed by virus-encoded proteinases. Despite their genetic austerity, picornaviruses have retained long 5' untranslated regions, which harbour cis-acting elements that promote initiation of translation independently of the uncapped 5' end of the viral messenger RNA. These elements are termed 'internal ribosomal entry sites' and are formed from highly structured RNA segments of at least 400 nucleotides. How these elements function is not known, but special RNA-binding proteins may be involved. The ribosome or its 40S subunit probably binds at or near a YnXmAUG motif (where Y is a pyrimidine and X is a purine) at the 3' border of the internal ribosomal entry site, which either provides the initiating codon or enables the ribosome to translocate to one downstream (E.W. et al., submitted). Initiation from most eukaryotic messenger RNAs usually occurs by ribosomal recognition of the 5' and subsequent scanning to the AUG codon. Here we describe a genetic strategy for the dissection of polyproteins which proves that an internal ribosomal entry site element can initiate translation independently of the 5' end.

Binding Sites↗

PubMed1991

Alternative translation initiation site in the DA strain of Theiler's murine encephalomyelitis virus.

Polyprotein processing studies of Theiler's murine encephalomyelitis virus (TMEV), a group of mouse picornaviruses, demonstrated synthesis of a protein we have called l during in vitro translations from the RNA of DA, a demyelinating strain of TMEV, but not GDVII, an acute neurovirulent strain. We have proposed that l is synthesized from an alternative initiation site in the DA leader (L) coding area out of phase with the polyprotein reading frame (R. P. Roos, W.-P. Kong, B. L. Semler, J. Virol. 63:5344-5353, 1989). We now provide support for this proposal from experiments involving in vitro translation of three separate mutations of an infectious DA cDNA clone: DA"l"-1, which contains a base mismatch at the putative initiation codon of l, DAL-1, which contains a base mismatch at the presumed authentic initiation site of L at the beginning of the polyprotein; and DAL:NheI, which contains nucleotides coding for a four-amino-acid insertion in the L coding area with a termination codon in the l reading frame. Our results demonstrate that the DA strain uses an alternative initiation site and reading frame to in vitro synthesize l. l may have a role in the biological activity of the virus.

Base Sequence↗

PubMed1993

Production of rat soluble and membrane-bound catechol O-methyltransferase forms from bifunctional mRNAs.

In the rat, the catechol O-methyltransferase (COMT) gene has been found to contain two promoters, P1 and P2. This organization enables the gene to produce a soluble (S-COMT) and a membrane-associated (MB-COMT) protein by using two in-frame ATG initiation codons (S- and MB-ATG). The P1 promoter expresses a 1.6 kb transcript (S-mRNA) which codes for the S-COMT polypeptide only. Here we demonstrate that the P2 promoter controls the expression of alternatively spliced 1.9 kb transcripts (MB-mRNA) which differ by a 27-nucleotide region immediately upstream of the MB-AUG codon. The presence of the 27-base sequence alters the nucleotide at position -3 from G to C, thereby changing the translation initiation context of the MB-AUG codon. Expression experiments in COS-7 cells using full-length COMT cDNAs showed that this alteration affected the initiation of the translation of the MB-AUG and consequently changed the relative amounts of MB- and S-COMT polypeptides produced. No proteolytic cleavage of the MB-COMT form to S-COMT was detected in in vitro or in vivo pulse-chase experiments. We conclude that the bifunctional 1.9 kb mRNAs are able to produce both S-COMT and MB-COMT polypeptide by the leaky scanning mechanism of translation initiation.

Alternative Splicing↗