Alternative translation initiation of Theiler's murine encephalomyelitis virus.
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DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) produce a chronic demyelinating disease in which the virus persists but has a restricted expression. We previously reported that TO subgroup strains, in addition to synthesizing the picornaviral polyprotein, use an alternative initiation codon just downstream from the polyprotein's AUG to translate an 18-kDa protein called L* that is out of frame with the polyprotein (H. H. Chen et al., Nat. Med. 1:927-931, 1995; W. P. Kong and R. P. Roos, J. Virol. 65:3395-3399, 1991). L* is critically important for virus persistence and the induction of the demyelinating disease (Chen et al., 1995; G. D. Ghadge et al. J. Virol. 72:8605-8612, 1998). We have proposed that variations in the amount of translation initiation from the L* AUG versus the polyprotein AUG may occur in different cell types and therefore affect the degree of expression of viral capsid proteins. We now demonstrate that ribosomal translation initiation at the polyprotein's initiation codon affects initiation at the L* AUG, suggesting that ribosomes land at the polyprotein's initiation codon before scanning downstream and initiating at the L* AUG. We also find that the viral 5' untranslated region affects utilization of the L* AUG. Surprisingly, mutant DA cDNAs were found to be infectious despite the presence of mutations of the polyprotein initiation codon or placement of a stop codon upstream of the L* AUG in the polyprotein's reading frame. Sequencing studies showed that these viruses had a second site mutation, converting the reading frame of L* into the polyprotein's reading frame; the results suggest that translation of the polyprotein during infection of these mutant viruses can be initiated at the L* AUG. These data are important in our understanding of translation initiation of TMEV and other RNAs that contain an internal ribosome entry site. (+info)
Analysis of base-pairing potentials between 16S rRNA and 5' UTR for translation initiation in various prokaryotes.
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MOTIVATION: It is well accepted that the 3' end of 16S rRNA is directly involved in prokaryotic translation initiation by pairing with the Shine-Dalgarno (SD) sequence, which is located in the ribosome-binding site of mRNA. According to Shine and Dalgarno, Escherichia coli 's 5' UTR has the pattern of 'AGGAGG' (SD sequence), which is complementary to the 3' end sequence of 16S rRNA. In this work, we systematically calculated free-energy values of the base pairing between the 3' end of 16S rRNA and the 5' UTR of mRNA, in order to analyze the base-pairing potentials in various prokaryotes. The free-energy values were then plotted over distances from the start codon to visualize the free-energy pattern of 5'UTRs. RESULTS: The average free-energy values fell sharply before the start codon in E. coli, which is consistent with the model that the 3' end of 16S rRNA base pairs with the SD sequence. Haemophilus influenzae, Bacillus subtilis and Helicobacter pylori show a similar pattern, suggesting that the organisms have basically the same mechanism of translation initiation as E. coli. Other eubacteria, such as Synechocystis PCC6803, Mycoplasma genitalium, Mycoplasma pneumoniae and Borrelia burgdorferi also show decreases in their free-energy values, although they are less evident. We also did the same analysis with a eukaryote genome as a control; no fall in free-energy values was observed between the 3' end of 18S rRNA and 5' UTRs of Saccharomyces cerevisiae, suggesting that this organism does not base pair in translation initiation. The three archaebacteria A. fulgidus, M. jannaschii and M. thermoautotrophicum show patterns similar to eubacteria, but not to S. cerevisiae, indicating that archaebacteria are closer to eubacteria than to eukaryotes with respect to the mechanism of translation initiation. From these observations, it appears that the shape of the curve produced by the algorithm can be used to predict the mechanism of translation initiation. AVAILABILITY: The C programs used in our analysis are available upon request. (+info)
A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA.
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Group II introns encode reverse transcriptases that promote RNA splicing (maturase activity) and then with the excised intron form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription (TPRT). Here, we show that the primary binding site for the maturase (LtrA) encoded by the Lactococcus lactis Ll.LtrB intron is within a region of intron domain IV that includes the start codon of the LtrA ORF. This binding is enhanced by other elements, particularly domain I and the EBS/IBS interactions, and helps position LtrA to initiate cDNA synthesis in the 3' exon as occurs during TPRT. Our results suggest how the maturase functions in RNA splicing and support the hypothesis that the reverse transcriptase coding region was derived from an independent genetic element that was inserted into a preexisting group II intron. (+info)
Multi-forms of human MTH1 polypeptides produced by alternative translation initiation and single nucleotide polymorphism.
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The human MTH1 gene for 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase, produces seven types (types 1, 2A, 2B, 3A, 3B, 4A and 4B) of mRNAs. The B-type mRNAs with exon 2b-2c segments have three additional in-frame AUGs in their 5' regions. We report here that these transcripts produce three forms of MTH1 polypeptides (p22, p21 and p18) in in vitro translation reactions. Three polypeptides were also detected in extracts of human cells, using western blotting. B-type mRNAs with a polymorphic alteration (GU-->GC) at the beginning of exon 2c that converts an in-frame UGA to CGA yielding another in-frame AUG further upstream, produced an additional polypeptide (p26) in vitro. Substitution of each AUG abolished the production of each corresponding polypeptide. Cell lines from individuals with the GC allele contain more B-type mRNAs than do those of GT homozygotes, and the former produce all of four polypeptides but the latter lack p26. Amounts of each polypeptide reflected copy number of the GC allele in each cell line. There is an apparent linkage dis-equilibrium between the two polymorphic sites, GT/GC at exon 2c and Val83/Met83 at codon 83 for p18. (+info)
Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli.
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Synthesis of the [NiFe] hydrogenases 1 and 2 of Escherichia coli is induced in response to anaerobiosis and is repressed when nitrate is present in the growth medium. The hydrogenase 1 and hydrogenase 2 enzymes are encoded by the polycistronic hyaABCDEF and hybOABCDEFG operons, respectively. Primer extension analysis was used to determine the initiation site of transcription of both operons. This permitted the construction of single-copy lacZ operon fusions, which were used to examine the transcriptional regulation of the two operons. Expression of both was induced by anaerobiosis and repressed by nitrate, which is in complete accord with earlier biochemical studies. Anaerobic induction of the hyb operon was only partially dependent on the FNR protein and, surprisingly, was enhanced by an arcA mutation. This latter result indicated that ArcA suppresses anaerobic hyb expression and that a further factor, which remains to be identified, is involved in controlling anaerobic induction of operon expression. Nitrate repression of hyb expression was mediated by the NarL/NarX and NarP/NarQ two-component regulatory systems. Remarkably, a narP mutant lacked anaerobic induction of hyb expression, even in the absence of added nitrate. Anaerobic induction of hya expression was dependent on the ArcA and AppY regulators, which confirms earlier observations by other authors. Nitrate repression of the hya operon was mediated by both NarL and NarP. Taken together, these data indicate that although the hya and hyb operons share common regulators, there are important differences in the control of expression of the individual operons. (+info)
Nucleosides as a carbon source in Bacillus subtilis: characterization of the drm-pupG operon.
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In Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized. The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase. The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here. When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity. Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon. Both enzymes are induced when nucleosides are present in the growth medium. Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate. Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression. Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells. The nucleoside-catabolizing system in B. subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source. When added alone, inosine barely supports growth of B. subtilis. This slow nucleoside catabolism contrasts with that of E. coli, which grows rapidly on a nucleoside as a carbon source. When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B. subtilis. The findings of this study therefore indicate that the B. subtilis system for nucleoside catabolism differs greatly from the well-studied system in E. coli. (+info)
Non-AUG initiation of AGAMOUS mRNA translation in Arabidopsis thaliana.
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The MADS box organ identity gene AGAMOUS (AG) controls several steps during Arabidopsis thaliana flower development. AG cDNA contains an open reading frame that lacks an ATG triplet to function as the translation initiation codon, and the actual amino terminus of the AG protein remains uncharacterized. We have considered the possibility that AG translation can be initiated at a non-AUG codon. Two possible non-AUG initiation codons, CUG and ACG, are present in the 5' region of AG mRNA preceding the highly conserved MADS box sequence. We prepared a series of AG genomic constructs in which these codons are mutated and assayed their activity in phenotypic rescue experiments by introducing them as transgenes into ag mutant plants. Alteration of the CTG codon to render it unsuitable for acting as a translation initiation site does not affect complementation of the ag-3 mutation in transgenic plants. However, a similar mutation of the downstream ACG codon prevents the rescue of the ag-3 mutant phenotype. Conversely, if an ATG is introduced immediately 5' to the disrupted ACG codon, the resulting construct fully complements the ag-3 mutation. The AG protein synthesized in vitro by initiating translation at the ACG position is active in DNA binding and is of the same size as the AG protein detected from floral tissues, whereas AG polypeptides with additional amino-terminal residues do not appear to bind DNA. These results indicate that translation of AG is initiated exclusively at an ACG codon and prove that non-AUG triplets may be efficiently used as the sole translation initiation site in some plant cellular mRNAs. (+info)
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.
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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. (+info)