Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe).
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The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated. 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions. At low oligonucleotide concentrations the binding occurs at the unique complementary site. At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides. In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide. The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement. (+info)
Identification of a ribonuclease H gene in both Mycoplasma genitalium and Mycoplasma pneumoniae by a new method for exhaustive identification of ORFs in the complete genome sequences.
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Exhaustive identification of open reading frames in complete genome sequences is a difficult task. It is possible that important genes are missed. In our efforts to reanalyze the intergenic regions of Mycoplasma genitalium and Mycoplasma pneumoniae, we have newly identified a number of new open reading frames (ORFs) in both M. genitalium and M. pneumoniae. The most significant identification was that of a ribonuclease H enzyme in both species which until now has not been identified or assumed absent and interpreted as such. In this paper we discuss the biological importance of RNase H and its evolutionary implication. We also stress the usefulness of our method for identifying new ORFs by reanalyzing intergenic regions of existing ORFs in complete genome sequences. (+info)
Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins.
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The testicular isoform of hormone-sensitive lipase (HSLtes) is encoded by a testis-specific exon and 9 exons common to the testis and adipocyte isoforms. In mouse, HSLtes mRNA appeared during spermiogenesis in round spermatids. Two constructs containing 1.4 and 0.5 kilobase pairs (kb) of the human HSLtes gene 5'-flanking region cloned upstream of the chloramphenicol acetyltransferase gene were microinjected into mouse oocytes. Analyses of enzyme activity in male and female transgenic mice showed that 0.5 kb of the HSLtes promoter was sufficient to direct expression only in testis. Cell transfection experiments showed that CREMtau, a testis-specific transcriptional activator, does not transactivate the HSLtes promoter. Using gel retardation assays, four testis-specific binding regions (TSBR) were identified using testis and liver nuclear extracts. The testis-specific protein binding on TSBR4 was selectively competed by a probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6 which are expressed in post-meiotic germ cells bound TSBR4. Mutation of the AACAAAG motif in TSBR4 abolished the binding. Moreover, binding of the high mobility group domain of Sox5 induced a bend within TSBR4. Together, our results showed that 0.5 kb of the human HSLtes promoter bind Sox proteins and contain cis-acting elements essential for the testis specificity of HSL. (+info)
Isolation of RNase H genes that are essential for growth of Bacillus subtilis 168.
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Two genes encoding functional RNase H (EC 3.1.26.4) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilis RNase HIII. The B. subtilis genome has an rnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB and rnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both the rnhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single rnhC homologue in the small genome of Mycoplasma species. (+info)
Initial recognition of U12-dependent introns requires both U11/5' splice-site and U12/branchpoint interactions.
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We have investigated the formation of prespliceosomal complex A in HeLa nuclear extracts on a splicing substrate containing an AT-AC (U12-type) intron from the P120 gene. Using an RNase H protection assay and specific blocking oligonucleotides, we find that recognition of the 5' splice-site (5'ss) and branchpoint sequence (BPS) elements by U11 and U12 snRNPs, respectively, displays strong cooperativity, requiring both sites in the pre-mRNA substrate for efficient complex formation. Deletion analysis indicates that beside the 5'ss and BPS, no additional elements in the pre-mRNA are necessary for A-complex formation, although 5' exon sequences provide stimulation. Cross-linking studies with pre-mRNAs containing the 5'ss or BPS alone indicate that recognition of the BPS by the U12 snRNP is stimulated at least 20- to 30-fold by the binding of the U11 snRNP to the 5'ss in the same pre-mRNA molecule, whereas recognition of the 5'ss by U11 is stimulated approximately fivefold by the U12/BPS interaction. These results argue that intron recognition in the U12-dependent splicing pathway is carried out by a single U11/U12 di-snRNP complex, suggesting greater rigidity in the intron recognition process than in the major spliceosome. (+info)
Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA.
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From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA. In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus. Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA. Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript. The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript. These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing. (+info)
UV-induced modifications in the peptidyl transferase loop of 23S rRNA dependent on binding of the streptogramin B antibiotic, pristinamycin IA.
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The naturally occurring streptogramin B antibiotic, pristinamycin IA, which inhibits peptide elongation, can produce two modifications in 23S rRNA when bound to the Escherichia coli 70S ribosome and irradiated at 365 nm. Both drug-induced effects map to highly conserved nucleotides within the functionally important peptidyl transferase loop of 23S rRNA at positions m2A2503/psi2504 and G2061/A2062. The modification yields are influenced strongly, and differentially, by P-site-bound tRNA and strongly by some of the peptidyl transferase antibiotics tested, with chloramphenicol producing a shift in the latter modification to A2062/C2063. Pristinamycin IA can also produce a modification on binding to deproteinized, mature 23S rRNA, at position U2500/C2501. The same modification occurs on an approximately 37-nt fragment, encompassing positions approximately 2496-2532 of the peptidyl transferase loop that was excised from the mature rRNA using RNAse H. In contrast, no antibiotic-induced effects were observed on in vitro T7 transcripts of full-length 23S rRNA, domain V, or on a fragment extending from positions approximately 2496-2566, which indicates that one or more posttranscriptional modifications within the sequence Cm-C-U-C-G-m2A-psi-G2505 are important for pristinamycin IA binding and/or the antibiotic-dependent modification of 23S rRNA. (+info)
DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes.
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In vitro selection was performed in a DNA library, made of oligonucleotides with a 30-nucleotide random sequence, to identify ligands of the human immunodeficiency virus type-1 trans-activation-responsive (TAR) RNA element. Aptamers, extracted after 15 rounds of selection-amplification, either from a classical library of sequences or from virtual combinatorial libraries, displayed an imperfect stem-loop structure and presented a consensus motif 5'ACTCCCAT in the apical loop. The six central bases of the consensus were complementary to the TAR apical region, giving rise to the formation of RNA-DNA kissing complexes, without disrupting the secondary structure of TAR. The RNA-DNA kissing complex was a poor substrate for Escherichia coli RNase H, likely due to steric and conformational constraints of the DNA/RNA heteroduplex. 2'-O-Methyl derivatives of a selected aptamer were binders of lower efficiency than the parent aptamer in contrast to regular sense/antisense hybrids, indicating that the RNA/DNA loop-loop region adopted a non-canonical heteroduplex structure. These results, which allowed the identification of a new type of complex, DNA-RNA kissing complex, demonstrate the interest of in vitro selection for identifying non-antisense oligonucleotide ligands of RNA structures that are of potential value for artificially modulating gene expression. (+info)