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PubMed2003

Production of recombinant thermostable proteins expressed in Escherichia coli: completion of protein synthesis is the bottleneck.

Heterologous expression and high yield purification of proteins are frequently required for structural and functional investigations. Purification of recombinant thermostable proteins is essentially trivial since unwanted mesophilic host protein can efficiently be removed by heat denaturation. However, heterologous expression in E. coli often results in truncated protein forms. In many cases, this is a consequence of abundant codons in heterologous genes, which are decoded by rare tRNAs in E. coli-a combination that can be responsible for translational stalling and termination during protein biosynthesis. Other complications may originate from potential initiation codons and ribosomal binding sites present inside the open reading frame of the target gene or from other less well defined phenomena such as mRNA instability. Separation of full-length protein from truncated forms is a serious chromatographic problem that can be solved in the expression step. We have investigated the heterologous expression and purification of two translation initiation factors from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus. Expression in E. coli was optimised to avoid truncated forms completely by complementation with the plasmids pSJS1244, pRIG, pCODON+ and pLysSR.A.R.E harbouring and expressing genes encoding rare tRNAs corresponding to the codons AGA, AGG, AUA, CUA, GGA, AAG and CCC. Two expression strains, C41(DE3) and C43(DE3) were found highly advantageous when combined with rare tRNA encoding plasmids as compared to BL21(DE3). We have also investigated the effects of site directed mutagenesis on rare lysine encoding AAG doublets as well as two methionine residues preceded by potential ribosomal binding sites. The expression approach presented here has enabled us to purify gram quantities of full-length protein by one step of ion-exchange chromatography and is generally applicable to many other heterologously expressed thermostable proteins.

Amino Acid Sequence↗
PubMed1986

Multiple upstream AUG codons mediate translational control of GCN4.

GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in yeast that is regulated at the translational level. The 5' leader of GCN4 mRNA contains four small open-reading-frames. By constructing point mutations in the initiation codons of these sequences, we show that they are essential for translational repression of GCN4. Each upstream AUG codon can repress translation; however, the two 3' proximal AUG codons are much more inhibitory than the 5' proximal AUG codons. Unexpectedly, the first AUG codon is required for efficient GCN4 expression under starvation conditions. This positive function appears to involve antagonism of the inhibitory effect of the 3' proximal AUG codons since it is dispensable in the absence of these sequences. The interaction between the upstream AUG codons is modulated by the trans-acting factors GCN2 and GCD1 in response to amino acid availability.

Amino Acids↗
PubMed1975

A possible role of the 5' terminal sequence of 16S ribosomal RNA in the recognition of initiation sequences for protein synthesis.

Extensive complementarity is found between the 5' end of 16S ribosomal RNA and protein synthesis initiation sites of bacteriophage RNA. Hybrids can be constructed from base sequences of 16S-RNA and two initiation regions on phage RNA. A model is proposed for the involvement of 16S-RNA in the unfolding of hairpin loops containing the initiation codon AUG.

Base Sequence↗
PubMed1983

[Study of the ribosome mRNA-binding region during different stages of translation. I. Functional activity of mRNA analogs, AUGU6 and its benzylidene derivatives, in ribosome-dependent protein synthesis].

Hexaribouridylic acid, prepared by digestion of poly(U) with cobra venom endonuclease, and trinucleotide AUG synthesized chemically by triester approach were joined by RNA-ligase to yield a nonaribonucleotide AUGU6 bearing the initiation codon at its 5'-terminus. 2',3'-O-(4-[N-(2-chloro(or hydroxy) ethyl-N-Methylamino])- benzylidene residues were introduced at the 3'-terminus of oligonucleotide AUGU6 and its benzylidene derivatives AUGU6CHRCl or AUGU6CHROH were obtained. The mRNA analogs synthesized were tested for their template activity in the formation of 70S initiation complex. AUGU6, AUGU6CHRC1 and AUGU6CHROH were shown to stimulate factor-dependent binding of fMet-tRNA to ribosomes. The effect of benzylidene fragment on the template activity of AUGU6CHROH in the course of translation process was studied. It was shown that AUGU6CHROH stimulates synthesis of di- and tripeptides with the same efficiency as AUGU6.

Benzylidene Compounds↗
PubMed1990

Scanning model for translational reinitiation in eubacteria.

Premature termination of translation in eubacteria, like Escherichia coli, often leads to reinitiation at nearby start codons. Restarts also occur in response to termination at the end of natural coding regions, where they serve to enforce translational coupling between adjacent cistrons. Here, we present a model in which the terminated but not released ribosome reaches neighboring initiation codons by lateral diffusion along the mRNA. The model is based on the finding that introduction of an additional start codon between the termination and the reinitiation site consistently obstructs ribosomes to reach the authentic restart site. Instead, the ribosome now begins protein synthesis at this newly introduced AUG codon. This ribosomal scanning-like movement is bidirectional, has a radius of action of more than 40 nucleotides in the model system used, and activates the first encountered restart site. The ribosomal reach in the upstream direction is less than in the downstream one, probably due to dislodging by elongating ribosomes. The proposed model has parallels with the scanning mechanism postulated for eukaryotic translational initiation and reinitiation.

Bacteriolysis↗
PubMed1987

Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity.

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoglycosides↗
PubMed1989

Antithrombin: structure, genomic organization, function and inherited deficiency.

Antithrombin is a major plasma protein inhibitor of proteinases generated during blood coagulation; it plays an important role in the regulation of thrombin in blood. The anticoagulant heparin greatly accelerates the rate of inactivation of proteinases by antithrombin, predominantly through its well defined, highly specific binding reaction with the inhibitor, but also through a less strictly defined interaction with some of the proteinases (such as thrombin). There is evidence for an analogous acceleratory mechanism in vivo, that functions by the binding of antithrombin to a subpopulation of heparan sulphate proteoglycans intercalated in the surface of endothelial cells. The location and structure of the gene for antithrombin are known. Both its overall organization and the structure of the subdomains of the expressed protein can be considered in terms of their relationships to a serine proteinase inhibitor superfamily, which is believed to have evolved from a common ancestor. The region of the antithrombin gene 5' to the coding region has been characterized. Unlike other members of the serpin family, there is no TATA-like promoter sequence. Two enhancer sequences have been identified that are homologous to enhancer regions of other genes. There are two polymorphisms: an intragenic polymorphism arising from a translationally silent A to G transition in codon 305, and a length polymorphism arising from the presence of 32 bp or 108 bp non-homologous sequences 345 bp upstream from the translation initiation codon. Inherited deficiency of antithrombin is associated with familial thromboembolism. The molecular genetic basis of some subtypes of deficiency is increasingly yielding to investigation. It is interesting to note that a number of mutations have been identified in CpG dinucleotides, supporting the suggestion that this dinucleotide sequence may represent a mutation hotspot in the human genome.

Amino Acid Sequence↗
PubMed1982

Nucleotide sequence of tobacco mosaic virus RNA.

Oligonucleotide primers have been used to generate a cDNA library covering the entire tobacco mosaic virus (TMV) RNA sequence. Analysis of these clones has enabled us to complete the viral RNA sequence and to study its variability within a viral population. The positive strand coding sequence starts 69 nucleotides from the 5' end with a reading frame for a protein of Mr 125,941 and terminates with UAG. Readthrough of this terminator would give rise to a protein of Mr 183,253. Overlapping the terminal five codons of this readthrough reading frame is a second reading frame coding for a protein of Mr 29,987. This gene terminates two nucleotides before the initiator codon of the coat protein gene. Potential signal sequences responsible for the capping and synthesis of the coat protein and Mr 29,987 protein mRNAs have been identified. Similar sequences within these reading frames may be used in the expression of sets of proteins that share COOH-terminal sequences.

Amino Acid Sequence↗
PubMed1991

Use of single-stranded DNA oligonucleotides in programming ribosomes for translation.

Single-stranded DNA (ssDNA) oligomers were compared to synthetic RNA oligomers in their ability to program E. coli ribosomes in vitro. AUG and dATG-containing oligomers promoted the non-enzymatic binding of fmet-tRNA to ribosomes, with similar dependence on time and magnesium concentration; only at 10 mM Mg++ or at low oligomer concentration was RNA slightly preferred in complex formation. These initiation complexes were biologically active in that fmet-tRNA, bound in response to ssDNA or RNA, was fully reactive with puromycin. While dAUG could not function as an initiation codon, p-dAUG functioned as well as AUG or dATG. However, dUAA and p-dUAA could not replace UAA in directing release-factor (RF) activity, and dTAA functioned only to a slight extent. Release factors had specificity for termination complexes containing dATGTAA, dATGTAG, or dATGTGA. At Mg++ concentrations of 15 mM or higher, these hexamers directed peptidyl transferase-dependent fmet-tRNA hydrolysis in the absence of RF. We suggest this RF-independent activation of peptidyl transferase as a unique system for studying the mechanism of termination. Overall, these results indicate that ssDNA can be used in place of RNA for certain studies of protein synthesis.

Codon↗
PubMed1982

Effect of secondary structure of messenger ribonucleic acid on the formation of initiation complexes with prokaryotic and eukaryotic ribosomes.

The effect of modification of the secondary structure of phage f2 RNA and brome mosaic virus (BMV) RNA 3 on the formation of initiation complexes in Escherichia coli and wheat germ protein-synthesizing systems was studied. Modification of the RNAs was achieved by using O-methylhydroxylamine, which specifically reacts with cytosines; this leaves the initiation codons unchanged and, under denaturing conditions, leads to irreversible unfolding of the RNA. E. coli ribosomes interact with newly exposed AUG/GUG codons in the modified templates forming polysomes, whereas they form monosomes with native f2 RNA or BMV RNA 3. With wheat germ ribosomes, disomes are formed in the presence of BMV RNA 3, either native or modified. With f2 RNA, eukaryotic ribosomes form monosomes, independent of the secondary structure of the template. The results indicate that, in contrast to prokaryotic ribosomes, binding of eukaryotic ribosomes to f2 RNA or BMV RNA 3 is not affected by modification of the secondary structure of these messengers.

Cells↗
PubMed1981

In vitro transcription of the yeast alcohol dehydrogenase I gene by homologous RNA polymerase B (II). Selective initiation and discontinuous elongation on a supercoiled template.

A new in vitro approach is used to investigate the specificity of purified yeast RNA polymerase B (II). The template is supercoiled, the transcription is primed by a dinucleotide, and the transcripts are analyzed by polyacrylamide gel electrophoresis after synthesis in the absence of one nucleoside triphosphate. Under these conditions, two recombinant plasmids carrying the gene or part of the gene for yeast alcohol dehydrogenase I direct the synthesis of a very limited number of oligonucleotides. Elongation of these prelabeled oligomers, using unlabeled substrates, occurs in a discontinuous way. A major transcript of 200 nucleotides accumulates transiently. Southern hybridization shows that it is initiated about 1,400 bases upstream from the origin of the yeast alcohol dehydrogenase I gene. A minor start was identified, by a modified runoff experiment, at position -35 from the AUG initiation codon. The location of this site is related to the presumptive in vivo transcription starts. The selectivity disappears when the template is a truncated DNA. Then, initiation occurs predominantly at nicks introduced by the restriction enzymes.

Alcohol Dehydrogenase↗
PubMed1995

Expression and characterization of proteins produced by mRNAs spliced into the X region of the human T-cell leukemia/lymphotropic virus type II.

In previous studies we showed that human T-cell leukemia/lymphotropic virus type I (HTLV-I) may produce novel proteins encoded in the X region. To investigate a possible correlation between expression of viral genes and different biologic properties of HTLV-I and HTLV-II, we analyzed expression of HTLV-II in the chronically infected cell line MoT. Reverse transcription-polymerase chain reaction analyses revealed that the virus produces several mRNAs singly or doubly spliced into the X region. Corresponding cDNAs were cloned and transfected into a HeLa cell line; resulting proteins were designated according to their sizes and coding open reading frames (ORFs). p10xI and p11xV were produced by a dicistronic doubly spliced mRNA. p10xI was generated by translation of the first exon of rex linked to the x-I ORF; p11xV was translated from the tax initiation codon linked to the x-V ORF. Two singly spliced polycistronic mRNAs produced p28xII, coded by the x-II ORF, and several isoforms generated by initiation within the x-III ORF. Studies of the proteins' subcellular localization revealed that they exhibited distinct targeting patterns. Comparison of these proteins with their HTLV-I counterparts indicated intriguing differences between these two viruses, suggesting that further study of the X region products may aid in defining genetic determinants of pathogenicity.

Alternative Splicing↗
PubMed2000

The structure and function of initiation factors in eukaryotic protein synthesis.

Protein synthesis is one of the most complex cellular processes, involving numerous translation components that interact in multiple sequential steps. The most complex stage in protein synthesis is the initiation process. It involves initiation factor-mediated assembly of a 40S ribosomal subunit and initiator tRNA into a 48S initiation complex at the initiation codon of an mRNA and subsequent joining of a 60S ribosomal subunit to form a translationally active 80S ribosome. The basal set of factors required for translation initiation has been determined, and biochemical, genetic, and structural studies are now beginning to reveal details of their individual functions in this process. The mechanism of translation initiation has also been found to be influenced significantly by structural properties of the 5' and 3' termini of individual mRNAs. This review describes some of the major developments in elucidating molecular details of the mechanism of initiation that have occurred over the last decade.

Amino Acid Sequence↗
PubMed1973

Discriminatory ribosome rebinding of isolated regions of protein synthesis initiation from the ribonucleic acid of bacteriophage R17.

To determine whether bacterial ribosomes recognize a distinguishing feature in the immediate vicinity of actual initiator codons or are directed to these sites through involvement of other portion(s) of the mRNA molecule, the interaction between ribosomes and defined (32)P-labeled initiator fragments from R17 RNA was studied. When incubated with mixtures of the three sites, ribosomes from Bacillus stearothermophilus (which initiate only the A protein on intact phage RNA) are able to select out the A fragment and discriminate against the coat and replicase initiator regions. By contrast, Escherichia coli ribosomes do not rebind that coat-protein region of R17 most efficiently, as they in the native RNA, but likewise prefer the A-protein initiator fragment. In both cases, ribosome binding of the isolated A site is comparable by several criteria to normal polypeptide-chain initiation on an intact R17 messenger RNA in vitro. E. coli ribosomal preference for the A site is confirmed in experiments with randomly fragmented R17 RNA, by both the initiation dipeptide and ribosome protection assay. Thus the A-protein ribosome-binding site of R17 RNA appears intrinsically to be a good initiator, while efficient recognition of the coat and replicase regions requires the participation of some portion of the remainder of the phage RNA molecule.

Bacillus↗
PubMed1984

Cloning and sequence analysis of cDNA for rat spleen thymosin beta 4.

Molecular cloning of a cDNA has established the sequence of the translated portion of the mRNA for rat spleen thymosin beta 4. The presence of a methionyl initiator codon immediately preceding the codon for the first seryl residue of mature thymosin beta 4 is consistent with previous results indicating the absence of a signal peptide in the product translated in vitro from rat spleen mRNA. The cDNA sequence analysis also established the presence of two terminator codons immediately following the codon for the COOH-terminal seryl residue. Thymosin beta 4 is thus synthesized as a 5100-dalton peptide containing 44 amino acid residues. Removal of the initiator methionyl residue and acetylation of the NH2-terminal serine residue would yield mature thymosin beta 4 containing 43 amino acids. The absence of a signal peptide makes it unlikely that thymosin beta 4 is a secreted peptide.

Amino Acid Sequence↗
PubMed1989

Structure and some characterization of the gene for phenylalanine ammonia-lyase from rice plants.

A nearly full-length cDNA and a genomic clone were isolated that encoded the phenylalanine ammonia-lyase (PAL) of rice plants, and the complete nucleotide sequences were determined. The gene encodes a polypeptide of 701 amino acid residues. The deduced amino acid sequence is highly similar to that of PAL from Phaseolus vulgaris deduced from an incomplete cDNA fragment. The cloned gene spans 4412 bp and consists of two exons and one intron. The site of initiation of transcription was located -86 nucleotides (position 1) upstream from the translational initiation codon by the primer-extension method. Sequences analogous to TATA-box and GC-box were found in the 5'-upstream region from the transcriptional initiation site. Southern blot analysis showed that the PAL gene of rice plants exists as a small multi-gene family. Within this family, the genomic clone isolated in this study was shown to be responsive by light. This also indicated that this genomic sequence functions as a gene for phenylalanine ammonia-lyase in rice plants in vivo.

Amino Acid Sequence↗
PubMed1990

Structure of colitis phage lytic enzyme gene.

A sequence of 163 amino acids was derived from the nucleotide sequence of a DNA fragment bearing the colitis phage lytic enzyme gene. The molecular weight, basicity and the sequence of the three N-terminal amino acids agreed with those of the phage lytic enzyme. The amino acids at the N-terminal of the colitis phage enzyme are homologous with those of T4, P22 and phi 29 phage lysozymes, to the extent of 18-34%. The transcription initiation site was mapped at 166 nucleotides upstream to the translation initiation codon, ATG. The Shine-Dalgarno sequence, the Pribnow box and the RNA polymerase recognition site were at 5.10 and 35 nucleotdies respectively, upstream to the transcription initiation site. The transcription termination signal, (the inverted repeat) and the termination site (the oligo TS) were at 7 and 28 nucleotides respectively, downstream to the translation termination site, TAA.

Amino Acid Sequence↗