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PubMed1998

Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis.

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

Alleles↗

PubMed1999

Characterization of two bifunctional Arabdopsis thaliana genes coding for mitochondrial and cytosolic forms of valyl-tRNA synthetase and threonyl-tRNA synthetase by alternative use of two in-frame AUGs.

We characterized two Arabidopsis thaliana cDNAs coding for class I valyl-tRNA synthetase and class II threonyl-tRNA synthetase. The proteins display characteristics of cytosolic enzymes, yet possess an N-terminal extension relative to their prokaryotic homologs. The proximal part of the N-terminal extension is a mitochondrial-targeting signal. Through transient expression of GFP fusions in tobacco cells, we demonstrated that both genes encode the cytosolic and mitochondrial forms of the enzymes by alternative use of two in-frame initiation codons. A long, mitochondrial form of the enzyme is translated from a first initiation codon at reduced levels because of a poor sequence context and a shorter, cytosolic form is translated from a second in-phase AUG, which is in a better context for translation initiation. Primer extension experiments revealed several transcript ends mapping upstream of the first AUG and between the two AUGs. Distal to the mitochondrial transit peptide both valyl-tRNA synthetase and threonyl tRNA synthetase possess an NH2-appended domain compared with their prokaryotic counterparts. This domain's amphiphilic helix is conserved between yeast and A. thaliana valyl-tRNA synthetase, suggesting an important role in translation. Based on the high structural similarities between yeast and A. thaliana valyl-tRNA synthetase, we propose that the acquisition of bifunctionality of valyl-tRNA synthetase predates the divergence of these two organisms.

Amino Acid Sequence↗

PubMed1995

Genomic mapping and sequence analysis of the fowl adenovirus serotype 10 hexon gene.

The gene for the major capsid protein (hexon) of fowl adenovirus serotype 10 (FAV-10) has been identified by the use of the expression vector pGEX and rabbit polyclonal antisera raised against FAV-10. The nucleotide sequence of the entire hexon gene has been determined. Sequence analysis revealed an open reading frame of 2808 bp coding for a putative polypeptide 936 amino acids long with a molecular mass of 105.5 kDa. The translation initiation codon has a local sequence which conforms with the optimal translation start sequence of CC(A/G)CCATGG. The location of the hexon gene in the FAV genome was from 46.85 to 52.81 map units, which is to the left of the hexon gene in the genomes of both bovine and human adenovirus (52.4 to 60.5 map units.). A splice acceptor site was identified 12 bp upstream of the initiation codon by using mRNA and PCR. It had the sequence TAGG which conforms to the consensus sequence of (C/T)AGG. Comparison of the amino acid sequence of the FAV-10 hexon with those of the bovine, human and murine hexon gene products revealed highest levels of identity occurring in the regions corresponding to the pedestals which form the base region of the hexon, and the lowest levels of identity in the regions corresponding to the loops which are exposed to the external environment.

Amino Acid Sequence↗

PubMed1995

The effect of changes in nucleotide sequence coding for the N-terminus on expression levels of ovine growth hormone variants in Escherichia coli.

The expression levels of coding sequences for pituitary growth hormone, introduced into Escherichia coli by genetic manipulation techniques, vary markedly according to the precise sequence introduced. In order to understand the basis of this variation more fully, we have studied the relationship between the level of expression in E. coli of a series of ovine growth hormone variants and the nucleotide sequences coding for their N-terminal regions. Sequence variation resulted from the introduction of deletions, or site-directed mutations, into a plasmid containing the coding sequence for ovine growth hormone preceded by the initiation codon and 25 bases derived from beta-galactosidase or linker regions of plasmid pUC8. The expression levels of the variants varied from less than 0.01% to over 34% of the total cell protein, indicating that changes in the nucleotide sequence close to the initiation codon had a marked effect on expression level. The results of a comparison of closely related sequences in pairs of plasmids giving poor or good expression are consistent with the hypothesis that poor translation of growth hormone mRNAs is caused by the presence of secondary structures close to the initiation codon. Secondary structures are identified that appear to explain the variation in expression levels.

Animals↗

PubMed2005

Polyamine transport by mammalian cells and mitochondria: role of antizyme and glycosaminoglycans.

The role of antizyme (AZ) and glycosaminoglycans in polyamine uptake by mammalian cells and mitochondria was examined using NIH3T3 and FM3A cells and rat liver mitochondria. AZ is synthesized as two isoforms (29 and 24.5 kDa) due to the existence of two initiation codon AUGs in the AZ mRNA. Most AZ existed as the 24.5-kDa form translatable from the second AUG, but a portion of the 29-kDa AZ from the first AUG was associated with mitochondria because of the presence of a mitochondrial targeting signal between the first and the second methionine. The predominance of the 24.5-kDa isoform was mainly due to the presence of spermidine and a favorable sequence context (Kozak sequence) at the second initiation codon AUG. Spermine uptake by NIH3T3 cells was inhibited by both 29- and 24.5-kDa AZs, but uptake by rat liver mitochondria was not influenced by either form of AZ. Because spermine uptake by mitochondria caused a release of cytochrome c, an enhancer of apoptosis, we looked for inhibitors of mitochondrial spermine uptake other than AZ. Cations such as Na+, K+, and Mg2+ were inhibitors of the mitochondrial uptake. It has been reported that heparan sulfate on glypican-1 plays important roles in spermine uptake by human embryonic lung fibroblasts. Heparin, but not heparan sulfate, slightly inhibited spermine uptake by FM3A cells in the absence of Mg2+ and Ca2+ but had no effect under physiological conditions in the presence of Mg2+ and Ca2+.

Amino Acid Sequence↗

PubMed2000

Methionine-independent initiation of translation in the capsid protein of an insect RNA virus.

Protein synthesis is believed to be initiated with the amino acid methionine because the AUG translation initiation codon of mRNAs is recognized by the anticodon of initiator methionine transfer RNA. A group of positive-stranded RNA viruses of insects, however, lacks an AUG translation initiation codon for their capsid protein gene, which is located at the downstream part of the genome. The capsid protein of one of these viruses, Plautia stali intestine virus, is synthesized by internal ribosome entry site-mediated translation. Here we report that methionine is not the initiating amino acid in the translation of the capsid protein in this virus. Its translation is initiated with glutamine encoded by a CAA codon that is the first codon of the capsid-coding region. The nucleotide sequence immediately upstream of the capsid-coding region interacts with a loop segment in the stem-loop structure located 15-43 nt upstream of the 5' end of the capsid-coding region. The pseudoknot structure formed by this base pair interaction is essential for translation of the capsid protein. This mechanism for translation initiation differs from the conventional one in that the initiation step controlled by the initiator methionine transfer RNA is not necessary.

Capsid↗

PubMed2004

Capped mRNA with a single nucleotide leader is optimally translated in a primitive eukaryote, Giardia lamblia.

The 5'-untranslated region (5'-UTR) of an mRNA plays an important role in translation initiation in eukaryotes. A minimal length of about 20 nucleotides is required to prevent leaky ribosome scanning. In one of the most primitive eukaryotes, Giardia lamblia, however, the mRNAs have 5'-UTRs mostly in the range of 0 to 14 nucleotides without a conserved sequence, which raises the question on how the ribosome could effectively scan such short 5'-UTRs for an accurate initiation of translation. In the present study, we expressed capped transcripts of luciferase gene in Giardia trophozoites via transfection and observed that when the 5'-UTR of the transcript was lengthened from 9 to 21 nucleotides, there was a corresponding decrease of translation efficiency. Conversely, shortening of the 5'-UTR from nine nucleotides down to a single nucleotide did not result in any reduced translation or leaky scanning. Translation appeared to initiate exclusively from the first initiation codon located downstream from the cap. Experimental evidence indicated also that a stem-loop structure immediately downstream from the initiation codon exerted significant inhibition on translation initiation when the 5'-UTR consisted of less than seven nucleotides. This inhibitory effect was abolished by increasing the distance between the stem-loop and the cap-G structure either upstream or downstream from the start codon, thus suggesting a spatial requirement for effective ribosome recruitment. Overall, our results suggest an absence of ribosome scanning for AUG in initiating translation in Giardia. A capped mRNA with a single nucleotide leader is apparently sufficient for recruiting ribosome and initiating translation.

5' Untranslated Regions↗

PubMed1994

Expression strategy of the M genome segment of Hantaan virus.

The medium (M) genome segment of Hantaan virus encodes the envelope glycoproteins, G1 and G2, in a continuous open reading frame with a gene order of 5'-G1-G2-3' with respect to the virus-complementary sense RNA. Because potential translation initiation codons and amino acids constituting typical signal sequences precede both the G1 and G2 genes, we sought to determine if G1 and G2 can be expressed independently. To investigate translational requirements for G1 and G2, we constructed M segment genes in which portions of the coding information were mutated or deleted, and transiently expressed these genes in eukaryotic cells by using a vaccinia virus/T7 RNA polymerase system. We found that G2 expression can occur by ribosomal access to the translation initiation codon preceding the G2 signal sequence (nucleotides 1934-1936), but that other upstream AUG codons cannot be used as efficiently. The presence of this codon, however, was not required for G2 expression because changing nucleotides 1934-1936 to CUG, GCG or AUG did not abrogate expression of G2. We also found that leaky ribosomal scanning, rather than internal initiation of translation was the most likely explanation for the observed independent translational initiation of G2, but that not all upstream, in-frame AUGs could serve as initiator codons. To assess the requirement for a continuous open reading frame for G1 and G2 expression, we expressed a gene which had G1 and G2 coding information in different reading frames. Although G1 was expressed at apparently normal levels, little or no G2 was expressed. In contrast, only G2 was expressed from a gene in which the carboxy-terminal G1 coding information was deleted and the remaining, truncated G1 was placed out of frame with respect to G2. These data suggest that reinitiation of translation may occur under some, but not all, circumstances when the polyprotein coding information is perturbed. Our results are consistent with biogenesis of G1 and G2 primarily or entirely according to the ribosomal scanning model.

Animals↗

PubMed1996

The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure.

The role of the RNA secondary structure in the 5' packaging signal region of human immunodeficiency virus type 1 (HIV-1) in initiating translation of gag mRNA has been investigated both in vitro and in the presence of cellular cofactors in vivo. Heat denaturation of the structure and mutagenic deletion both lead to an increase in levels of translated products, indicating that the structure is a significant inhibitor of translation. The proximity of the gag AUG to the packaging signal structure suggested that it might function as an internal ribosome entry site. However, in both a cell-free system and eukaryotic cells, translation will initiate at a novel upstream initiation codon introduced within the 5' noncoding region. This codon is utilized exclusively, resulting in gag protein products with an extra 11 amino acids at the amino terminus, which, when expressed in T lymphocytes, are confined intracellularly, probably because of the lack of an N-terminal glycine myristoylation signal. Deletion of the secondary structure abolishes gag production even in the presence of tat and rev in trans. Using dicistronic constructs containing the HIV-1 5' leader cloned between two heterologous open reading frames, we were unable to detect any significant expression of the second open reading frame that would have been supportive of an internal ribosome entry site mechanism. Using mutant proviruses either lacking the entire packaging signal structure region or containing the introduced upstream initiation codon in long-term replication studies, we were unable to detect reverse transcriptase activity in culture supernatants. The 5' packaging signal structure of HIV-1 does not serve as an internal ribosome entry site. The translation of gag is consistent with ribosomal scanning. However, the packaging signal structure causes significant translational inhibition.

Amino Acid Sequence↗

PubMed2004

Mutation and selection on the anticodon of tRNA genes in vertebrate mitochondrial genomes.

The H-strand of vertebrate mitochondrial DNA is left single-stranded for hours during the slow DNA replication. This facilitates C-->U mutations on the H-strand (and consequently G-->A mutations on the L-strand) via spontaneous deamination which occurs much more frequently on single-stranded than on double-stranded DNA. For the 12 coding sequences (CDS) collinear with the L-strand, NNY synonymous codon families (where N stands for any of the four nucleotides and Y stands for either C or U) end mostly with C, and NNR and NNN codon families (where R stands for either A or G) end mostly with A. For the lone ND6 gene on the other strand, the codon bias is the opposite, with NNY codon families ending mostly with U and NNR and NNN codon families ending mostly with G. These patterns are consistent with the strand-specific mutation bias. The codon usage biased towards C-ending and A-ending in the 12 CDS sequences affects the codon-anticodon adaptation. The wobble site of the anticodon is always G for NNY codon families dominated by C-ending codons and U for NNR and NNN codon families dominated by A-ending codons. The only, but consistent, exception is the anticodon of tRNA-Met which consistently has a 5'-CAU-3' anticodon base-pairing with the AUG codon (the translation initiation codon) instead of the more frequent AUA. The observed CAU anticodon (matching AUG) would increase the rate of translation initiation but would reduce the rate of peptide elongation because most methionine codons are AUA, whereas the unobserved UAU anticodon (matching AUA) would increase the elongation rate at the cost of translation initiation rate. The consistent CAU anticodon in tRNA-Met suggests the importance of maximizing the rate of translation initiation.

Animals↗

PubMedDate not supplied

Translational selection in the expression of the hepatitis B virus envelope proteins.

The region of the hepatitis B virus (HBV) genome coding for the viral envelope proteins contains three inphase ATGs that are conserved among viral subtypes. Each of these ATGs can be used as mRNA initiation codons. The three translated proteins share a carboxy-terminal region (the S protein) and extend amino-terminally to include the pre-S2 region in the middle (M) protein, and the pre-S1 and pre-S2 regions in the large (L) protein. We have inserted the HBV DNA coding for the M protein into a baculovirus expression vector. Infected insect cells transcribe a mRNA that is initiated solely within a baculovirus promoter, and that contains the initiator codons for both M and S proteins. Although these cells primarily secrete the M protein, the major translational product is the S protein, which is not secreted. This preferential translation, the result of the use of an internal initiator codon, demonstrates that the regulation of HBV envelope protein production can occur at the translational level.

Cloning, Molecular↗

PubMed1987

Regulation, initiation, and termination of the cenA and cex transcripts of Cellulomonas fimi.

We characterized the in vivo transcripts of two Cellulomonas fimi genes, the cenA gene, which encodes an extracellular endo-beta-1,4-glucanase (EC 3.2.1.4) and the cex gene, which encodes an extracellular exo-beta-1,4-glucanase (EC 3.2.1.91). By Northern blot analysis, cenA mRNA was detected in C. fimi RNA preparations from glycerol- and carboxymethyl cellulose-grown cells but not from glucose-grown cells. In contrast, cex mRNA was detected only in the preparations from carboxymethyl cellulose-grown cells. Therefore, the transcription of these genes is subject to regulation by the carbon source provided to C. fimi. By nuclease S1 protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, 5' termini were found 51 and 62 bases before the cenA translational initiation codon and 28 bases before the cex translational initiation codon. S1 mapping with unlabeled DNA probes and C. fimi RNA which had been isolated in vivo but which had been 5' labeled in vitro with guanylyltransferase and [alpha-32P]GTP confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. Comparative analysis of the DNA sequences immediately upstream of the initiation sites of the cenA and cex mRNAs revealed a 30-base-pair region where these two sequences display at least 66% homology. S1 mapping was also used to locate the 3' termini of the cenA and cex transcripts. Three 3' termini were found for cenA messages, whereas only one 3' terminus was identified for cex mRNA. The transcripts of both genes terminate in regions where their corresponding DNA sequences contain inverted repeats.

Bacteria, Anaerobic↗

PubMed1994

Control of ColE2 plasmid replication: negative regulation of the expression of the plasmid-specified initiator protein, Rep, at a posttranscriptional step.

The incA gene of ColE2 is involved in the copy number control and incompatibility. Two promoters were identified around the incA gene. Transcription of the mRNA for the essential plasmid-coded initiator protein (Rep) mainly starts at a site about 140 bp upstream of the initiation codon of the Rep protein. The second transcript (RNA I) of about 115 nucleotides with two stem-and-loop structures is entirely complementary to the 5' untranslated region of the Rep mRNA. By using translational and transcriptional fusions of the rep gene of ColE2 and the lacZ gene of Escherichia coli, the incA gene product was shown to regulate expression of the rep gene at a posttranscriptional step. The results also suggest that the target of the incA gene product is the 5' untranslated region of the Rep mRNA. Deletion analyses reported here show that a region(s) about 17 to 70 bp upstream of the initiation codon of the Rep protein and another region inside the coding frame are important for efficient production of the Rep protein. This suggests that some additional sequence elements other than the initiation codon and the Shine-Dalgarno region and/or a secondary structure of the Rep mRNA are required for efficient production of the Rep protein. These results show that RNA I is an antisense RNA for the Rep mRNA and imply that it might regulate expression of the rep gene at the initiation step of translation by sequestering such additional sequence elements and/or by disrupting RNA secondary structure. We propose that RNA I represents the incA gene product.

Bacterial Proteins↗

PubMed1988

Mutations at a Zn(II) finger motif in the yeast eIF-2 beta gene alter ribosomal start-site selection during the scanning process.

We have genetically reverted HIS4 initiator codon mutants in yeast and identified three unlinked genes, sui1, sui2, and SUI3 (suppressors of initiator codon mutants), which when mutated confer the ability to initiate at HIS4 despite the absence of an AUG start codon. Molecular and biochemical characterization shows that SUI3 encodes the beta-subunit of the eukaryotic translation initiation factor eIF-2. SUI3 suppressor genes contain single base changes at a Zn(II) finger motif. This motif is present in a cDNA sequence encoding the human eIF-2 beta gene product. Mutations in SUI3 suppressor alleles change amino acids that are conserved in the yeast and human motifs. Protein sequence analysis shows that a mutant beta-subunit allows initiation at a UUG codon in the absence of an AUG start codon at HIS4. Taken together, these data implicate a nucleic acid-binding domain of eIF-2 as an important component of the "scanning" ribosome that participates in recognition of a start codon.

Alleles↗

PubMed1989

Yeast translation initiation suppressor sui2 encodes the alpha subunit of eukaryotic initiation factor 2 and shares sequence identity with the human alpha subunit.

Genetic reversion of HIS4 initiator codon mutations in yeast has identified three unlinked genes, sui1, sui2, and SUI3 (suppressors of initiator codon mutations), which when mutated confer the ability to initiate translation at HIS4 despite the absence of an AUG start codon. We have previously demonstrated that the SUI3 gene encodes the beta subunit of the eukaryotic initiation factor 2 (eIF-2) and that mutations at a Zn(II) finger motif of SUI3 alter the start site selection process in yeast. In this report, molecular and biochemical characterizations show that the sui2 suppressor gene encodes the alpha subunit of eIF-2. The amino acid sequence of sui2 is 58% homologous to that encoded by the cDNA of the human eIF-2 alpha. Mutations in the sui2 suppressor alleles occur in the amino-terminal portion of the protein and change amino acids that are identical at the same relative position in the yeast and human proteins. Protein sequence analysis shows that a sui2 mutant yeast strain allows initiation at a UUG codon in the absence of an AUG codon at HIS4. These data further suggest that eIF-2 is an important component of the preinitiation complex that mediates ribosomal recognition of a start codon during the scanning process.

Amino Acid Sequence↗

PubMed2004

Expression of the two nested overlapping reading frames of turnip yellow mosaic virus RNA is enhanced by a 5' cap and by 5' and 3' viral sequences.

The translation efficiency of an mRNA molecule is typically determined by its 5'- and/or 3'-untranslated regions (UTRs). Previously, we have found that the 3'-UTR of Turnip yellow mosaic virus (TYMV) RNA enhances translation synergistically with a 5' cap. Here, we use a luciferase reporter system in cowpea protoplasts to show that the 5' 217 nucleotides from TYMV genomic RNA enhance expression relative to a vector-derived 17-nucleotide 5'-UTR. Maximum expression was observed from RNAs with a cap and both 5' and 3' TYMV sequences. In paired reporter constructs, the 5' 217 nucleotides harboring the UTR and the first 43 or 41 codons of the two overlapping TYMV open reading frames (ORFs), ORF-69 and ORF-206, respectively, were fused in frame with the luciferase gene. This allowed expression from the initiation codon of each ORF (AUG69 and AUG206) to be monitored separately but from the normal sequence environment. Expression from both AUG codons was heavily dependent on a 5' cap, with a threefold-higher expression occurring from AUG69 than from AUG206 in the presence of the genomic 3'-UTR. Changes that interrupted the cap/3'-UTR synergy (i.e., removal of the cap or TYMV 3'-UTR) resulted in a higher proportion of initiation from AUG206. Mutation of the 3'-UTR to prevent aminoacylation, as well as deletion of 75% of the 5'-UTR, likewise resulted in a lower ratio of expression from AUG69 relative to AUG206. Mutation of each AUG initiation codon increased initiation from the other. Taken together, these results do not fully conform to the expectations of standard leaky ribosomal scanning and leave open the precise mechanism of ribosome commitment to AUG69 and AUG206. However, our observations do not support a recent proposal based on in vitro studies in which the 3'-UTR is proposed to direct cap-independent initiation specifically at AUG206 and not at AUG69 (S. Barends et al., Cell 112:123-129, 2003).

3' Untranslated Regions↗

PubMed1992

Organization of ATPA coding and 3' flanking sequences associated with cytoplasmic male sterility in Phaseolus vulgaris L.

A region of the mitochondrial genome associated with cytoplasmic male sterility (CMS) in Phaseolus vulgaris was flanked by two different repeated sequences designated x and y. The DNA sequence of the CMS-unique region and a portion of each flanking repeat was determined. Repeat x contained a complete coding copy of the F1 ATPase subunit A (atpA) gene, as well as an open reading frame (orf) predicting a protein of 209 amino acids. The TGA termination codon of the atpA gene and the ATG initiation codon of orf209 were overlapping. These reading frames were oriented with their 3' ends proximal to the CMS-unique region. The CMS-unique region of 3736 nucleotides contained numerous orfs. The longest of these predicted proteins being of 239, 98 and 97 amino acids. The 3' coding and 3' flanking regions of orf98 were derived from an internal region of the higher plant chloroplast tRNA alanine intron. The region of repeat y immediately adjacent to the CMS-unique region contained the 111 carboxy-terminal coding residues of the apocytochrome b (cob) gene. This segment was oriented with its 5' end proximal to the CMS-unique region, but cob gene sequences were not fused to an initiation codon within the unique region.

Amino Acid Sequence↗

PubMed2003

Rapid detection of six common Mediterranean and three non-Mediterranean alpha-thalassemia point mutations by reverse dot blot analysis.

We describe the implementation of reverse dot blot (RDB) hybridization as a rapid nonradioactive method for the identification of six frequent globin gene point mutations in the Mediterranean population: alpha(Hph)alpha: alpha2 IVS I donor site GGTGAGG --> GG-----; alpha(NcoI)alpha: alpha2 initiation codon ATG --> ACG; alpha(TSaudi)alpha: alpha2Poly A signal AATAA --> AATAAG; alpha(Icaria)alpha: alpha2 termination codon TAA --> AAA (Ter --> LYS); alpha(CS)alpha: alpha2 termination codon TAA --> CAA (Ter --> gly); alphaalpha(NcoI): alpha1 initiation codon ATG --> GTG; and three alpha2 globin gene point mutations found in immigrants in Italy: alpha(T-Quongsze)alpha: alpha2 codon 12 CTG --> CCG (Leu --> Pro); alpha(Seal Rock)alpha: alpha2 termination codon TAA --> GAA (TER --> GLU); and alpha(Koyadora)alpha: alpha2 termination codon TAA --> TCA (TER --> SER). The method uses the principle of allele-specific oligonucleotide (ASO) hybridization, but it is a nonradioactive method and permits rapid and simultaneous typing of point mutations and small deletions.

Alleles↗