Myeloid cell-lineage and premylocytic-stage-specific- expression of themouse myeloperoxidase gene is controlled at initiation as well as elongation levels of transcription.
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The myeloperoxidase (MPO) is an important microbicidal protein present at high concentration in the primary granule of mature granulocyte and its expression is regulated in both myeloidcell-lineage and premyelocytic-stage-specific manners. A better understanding of the underlying control mechanisms should provide insights into the temporal and co-ordinate regulation of the gene expression during granulopoiesis. We have identified its promoter by mapping the start(s) of transcription using various molecular approaches together with demonstrating the promoter function of the relevant DNA segment in a transient transfection reporter assay. Besides the major start of transcription mapped at G residue, 11 nucleotide upstream of the 3' end of exon 0, the usage of that is specific to the MPO expressing cell lines, we have shown that irrespective of the MPO-expression status of the hematopoietic cells, transcription occurs broadly within a two kb region upstream of the 5' proximity of the gene, and is largely terminated in intron 2. These data support a model of the premyelocytic-stage-specific MPO expression, the control of which is operated at initiation as well as elongation levels of transcription. (+info)
Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA). Involvement of a structural change of the Shine-Dalgarno sequence and the initiation codon aug in oppa mRNA.
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We previously suggested that the degree of polyamine stimulation of oligopeptide-binding protein (OppA) synthesis is dependent on the secondary structure and position of the Shine-Dalgarno (SD) sequence of OppA mRNA. To study the structural change of OppA mRNA induced by polyamines and polyamine stimulation of initiation complex formation, four different 130-mer OppA mRNAs containing the initiation region were synthesized in vitro. The structural change of these mRNAs induced by polyamines was examined by measuring their sensitivity to RNase T(1), specific for single-stranded RNA, and RNase V(1), which recognizes double-stranded or stacked RNA. In parallel, the effect of spermidine on mRNA-dependent fMet-tRNA binding to ribosomes was examined. Our results indicate that the secondary structure of the SD sequence and initiation codon AUG is important for the efficiency of initiation complex formation and that spermidine relaxes the structure of the SD sequence and the initiation codon AUG. The existence of a GC-rich double-stranded region close to the SD sequence is important for spermidine stimulation of fMet-tRNA binding to ribosomes. Spermidine apparently binds to this GC-rich stem and causes a structural change of the SD sequence and the initiation codon, facilitating an interaction with 30 S ribosomal subunits. (+info)
Translation initiation factor eIF4B interacts with a picornavirus internal ribosome entry site in both 48S and 80S initiation complexes independently of initiator AUG location.
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Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation. (+info)
Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis.
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We analysed the Bacillus subtilis protein coding sequences termini, and compared it to other genomes. The analysis focused on signals, com-positional biases of nucleotides, oligonucleotides, codons and amino acids and mRNA secondary structure. AUG is the preferred start codon in all genomes, independent of their G+C content, and seems to induce less stable mRNA structures. However, it is not conserved between homologous genes neither is it preferred in highly expressed genes. In B.subtilis the ribosome binding site is very strong. We found that downstream boxes do not seem to exist either in Escherichia coli or in B.subtilis. UAA stop codon usage is correlated with the G+C content and is strongly selected in highly expressed genes. We found less stable mRNA structures at both termini, which we related to mRNA-ribosome and mRNA-release-factor interactions. This pattern seems to impose a peculiar A-rich nucleotide and codon usage bias in these regions. Finally the analysis of all proteins from B.subtilis revealed a similar amino acid bias near both termini of proteins consisting of over-representation of hydrophilic residues. This bias near the stop codon is partially release-factor specific. (+info)
Bacterial start site prediction.
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With the growing number of completely sequenced bacterial genes, accurate gene prediction in bacterial genomes remains an important problem. Although the existing tools predict genes in bacterial genomes with high overall accuracy, their ability to pinpoint the translation start site remains unsatisfactory. In this paper, we present a novel approach to bacterial start site prediction that takes into account multiple features of a potential start site, viz., ribosome binding site (RBS) binding energy, distance of the RBS from the start codon, distance from the beginning of the maximal ORF to the start codon, the start codon itself and the coding/non-coding potential around the start site. Mixed integer programing was used to optimize the discriminatory system. The accuracy of this approach is up to 90%, compared to 70%, using the most common tools in fully automated mode (that is, without expert human post-processing of results). The approach is evaluated using Bacillus subtilis, Escherichia coli and Pyrococcus furiosus. These three genomes cover a broad spectrum of bacterial genomes, since B.subtilis is a Gram-positive bacterium, E.coli is a Gram-negative bacterium and P. furiosus is an archaebacterium. A significant problem is generating a set of 'true' start sites for algorithm training, in the absence of experimental work. We found that sequence conservation between P. furiosus and the related Pyrococcus horikoshii clearly delimited the gene start in many cases, providing a sufficient training set. (+info)
'Primer alignment-and-extension': a novel mechanism of viral RNA recombination responsible for the rescue of inactivated poliovirus cDNA clones.
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In the course of experiments designed to assess the potential role of alternative open reading frames (ORF) present in the 5'-terminal untranslated region (5'-UTR) of poliovirus type 1 (Mahoney strain) genomic RNA, we came across a double mutation that completely abrogated the infectivity of full-length cDNA clones. The infectivity was rescued in trans by cotransfecting COS-1 cells with short RNA transcripts of the wild-type 5'-UTR of poliovirus type 2 Lansing, provided a free 3'-OH was available. Direct sequencing of the viral RNA revealed that the infectious viruses recovered were recombinants Lansing/Mahoney, with variable points of 'crossing-over'. A novel mechanism of RNA-RNA recombination, which we propose to call 'primer alignment-and-extension', is described that would explain the high rate of recombination of RNA viruses observed in natural conditions. (+info)
Repression of IS200 transposase synthesis by RNA secondary structures.
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The IS 200 transposase, a 16 kDa polypeptide encoded by the single open reading frame (ORF) of the insertion element, has been identified using an expression system based on T7 RNA polymerase. In wild-type IS 200, two sets of internal inverted repeats that generate RNA secondary structures provide two independent mechanisms for repression of transposase synthesis. The inverted repeat located near the left end of IS 200 is a transcriptional terminator that terminates read-through transcripts before they reach the IS 200 ORF. The terminator is functional in both directions and may terminate >80% of transcripts. Another control operates at the translational level: transposase synthesis is inhibited by occlusion of the ribosome-binding site (RBS) of the IS 200 ORF. The RBS (5'-AGGGG-3') is occluded by formation of a mRNA stem-loop structure whose 3' end is located only 3 nt upstream of the start codon. This mechanism reduces transposase synthesis approximately 10-fold. Primer extension experiments with AMV reverse transcriptase have provided evidence that this stem-loop RNA structure is actually formed. Tight repression of transposase synthesis, achieved through synergistic mechanisms of negative control, may explain the unusually low transposition frequency of IS 200. (+info)
A sequence downstream of the initiation codon is essential for cold shock induction of cspB of Escherichia coli.
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Cold shock induction of cspB has been shown to be primarily regulated at the mRNA level. Here, we demonstrate that the induction of cspB at low temperature also requires the translational cis-acting element called the downstream box (DB). Full induction of cspB at low temperature is achieved in the presence of both the Shine-Dalgarno sequence and DB. We propose that the DB sequence functions as a translational enhancer for the biosynthesis of CspB to bypass the inhibitory effect in translation caused by cold shock. (+info)