Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus. The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons. The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping. The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina. These proteins shared nearly 85% homology. A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms. Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M. racemosus. Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA.
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PMID:The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus. 302 62

A cloned DNA segment from Bacillus subtilis containing 21 tRNA genes was introduced into Escherichia coli. In the B. subtilis genome, these tRNA genes are located after an rRNA gene set and before tandem terminators. The rRNA and tRNA genes are thought to represent a single transcriptional unit. However, another putative promoter occurs after the second tRNA gene within the tRNA gene cluster and has a sequence compatible with both the major B. subtilis (sigma 43 type) promoter and the major E. coli promoter. The B. subtilis 21-tRNA-gene cluster was introduced into E. coli to see whether this promoter would be recognized in E. coli, to determine the start point of transcription in the E. coli system, and to see whether mature B. subtilis tRNAs would be transcribed and processed in E. coli. Expression was evaluated by monitoring levels of aminoacylation of mature tRNAs extracted from E. coli containing plasmids with or without the B. subtilis tRNA genes and by examining profiles of isoaccepting species on columns of RPC-5. S1 nuclease mapping was performed to define the starting point for transcription. The results indicated that a putative promoter located within the B. subtilis tRNA gene region was functional when cloned into E. coli and that it initiated at the same nucleotide as it does in B. subtilis. In addition, at least some B. subtilis tRNA genes could be transcribed and processed in E. coli to mature tRNAs capable of accepting an amino acid.
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PMID:Expression in Escherichia coli of Bacillus subtilis tRNA genes from a promoter within the tRNA gene region. 308 55

We succeeded in identifying a promoter element within 200 base pairs upstream a transcriptional unit comprising only a 23S rRNA, 5S rRNA and a tRNA(gly) gene in Thermus thermophilus HB8 [1, 2]. This element shows a high degree of homology to the -35 and -10 consensus sequences for promoters described for Escherichia coli [3, 4]. The promoter activity was measured by the induction of the synthesis of functional chloramphenicol acetyltransferase in Escherichia coli. A region located at the transcriptional start, rich in guanosines and cytidines, is very similar in sequence to the one believed to be under stringent control in stable RNA and ribosomal protein genes of Escherichia coli [5]. Employing nuclease S1 protection we were able to determine the in vivo start of transcription, which was identical with the in vitro start using Escherichia coli RNA-polymerase. Furthermore we identified sequences in the region following the origin of transcription, which are homologous to sections in Escherichia coli rrn promoter-leader regions responsible for antitermination. Our finding of a promoter immediately preceding a 23S/5S rRNA operon proves a transcriptional decoupling of the 16S rRNA genes, a situation so far unprecedented among prokaryotes.
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PMID:An unusual rRNA operon constellation: in Thermus thermophilus HB8 the 23S/5S rRNA operon is a separate entity from the 16S rRNA operon. 312 27

This is part of a series of two papers on gene regulation in Bacillus subtilis rRNA-tRNA operons that contain large clusters of tRNA genes. The preceding paper (Vold, B.S., Okamoto, K., Murphy, B.J., and Green, C.J. (1988) J. Biol. Chem. 263, 14480-14484) investigates the rrnB operon containing 21 tRNA genes, and this paper investigates a B. subtilis rRNA-tRNA operon containing 16 tRNA genes and a minor 5 S rRNA. Hybridization studies suggest this minor 5 S rRNA occurs as a single copy in the B. subtilis 168 genome. S1 nuclease mapping indicates that this minor 5 S rRNA gene has its own promoter. No promoters have been found immediately 5' to any of the major 5 S rRNA species in B. subtilis rRNA operons. S1 mapping of the spacer region between the 23 S and minor 5 S rRNA revealed that the maturation of the 23 S rRNA in this operon may arise from an unusual processing mechanism. S1 nuclease mapping experiments suggest the existence of a promoter element immediately upstream of the last gene, for tRNA(Leu CAA), in the operon. A precursor leucine tRNA resulting from transcription of this last tRNA gene was observed in Northern hybridizations, and the amounts of this precursor increased during sporulation. A single terminator-like element is located just upstream of this last tRNA gene; however, S1 nuclease mapping experiments suggest that some read-through transcription occurs. Thus, all 16 tRNA genes are under control of the upstream 16 S rRNA promoters and the minor 5 S rRNA promoter. However, the last tRNA gene is primarily under the control of its own unique promoter.
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PMID:Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons. II. Unique properties of an operon containing a minor 5 S rRNA gene. 313 57

Although the sequence and organization of many Bacillus subtilis tRNA genes are known, primary transcripts from these regions have not been previously analyzed. In this paper, S1 nuclease mapping, S1-type mapping, and Northern analyses were applied to the end of the 23 S rRNA, the 5 S rRNA, and the 21 tRNA genes of B. subtilis operon rrnB. Primary transcripts from the 5 S rRNA and tRNA genes up to approximately 600-800 nucleotides long were observed with S1-type mapping. The presence of discrete bands of processing intermediates indicated preferred processing points within the initial transcript. S1 nuclease mapping delineated a start point for transcription between the second and third tRNA genes. The -10 sequence was within the 37-base pair spacer region between tRNA genes, and the -35 sequence was within the structural gene for the upstream tRNA. Precursors from this region were evident during midexponential growth and two sporulation stages. Thus, in addition to promotion from the rRNA promoters, 19 of the 21 downstream tRNA genes are also under the control of an internal tRNA gene promoter. The accompanying paper (Vold, B. S., Green, C. J., Narasimhan, N., Strem, M., and Hansen, J. N. (1988) J. Biol. Chem. 263, 14485-14490) investigates the minor 5 S rRNA and 16 tRNA genes of another rRNA-tRNA gene set and emphasizes unique promoter elements in that system as well as a potentially unique rRNA processing scheme.
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PMID:Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons. I. The tRNA gene cluster of rrnB has an internal promoter. 317 May 52

Expression of 5 yeast mitochondrial tRNA genes (Ala, Ile, Tyr, Asn and Metm), localized upstream from the oxil gene has been analyzed by in vitro capping using guanylyltransferase, northern hybridization and S1 nuclease mapping in the wild type and a rho-strain. The 5 tRNA sequences belong to the same transcriptional unit which is initiated 133 bp upstream from the tRNA(Ala) gene at a promoter sequence TTATAAGTA. Furthermore, a truncated tRNA(Tyr) transcript, 2 nucleotides shorter than mature tRNA(Tyr) has been found, only in the rho-strain. This minor transcript may result from secondary transcription initiation at a variant nonanucleotide sequence, ATATAAGGA, which overlaps the tRNA(Tyr) coding sequence by 3 nucleotides. The polycistronic precursor has proven to be useful in investigation of the mechanisms of tRNA processing. Maturation of this primary transcript proceeds exclusively by precise endonucleolytic cleavages at the 5' and 3'-ends of tRNA sequences.
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PMID:Transcription initiation and RNA processing of a yeast mitochondrial tRNA gene cluster. 330 93

The eucaryotic elongation factor Tu (eEF-Tu) is a single polypeptide with an approximate Mr of 53,000. During protein synthesis eEF-Tu promotes the binding of aminoacyl-tRNA to the ribosome. To study the expression of the gene(s) for this factor, a genomic clone was isolated that contains a mouse eEF-Tu gene. We screened a phage genomic library with a synthetic oligonucleotide probe complementary to a region of the Saccharomyces cerevisiae and Artemia sp. eEF-Tu genes which codes for an area that is highly conserved between both yeast and Artemia sp. eEF-Tu. From approximately 75,000 phage plaques we obtained five isolates with apparently identical inserts. All five clones contained a 3.8-kilobase EcoRI fragment that hybridized to additional oligonucleotide probes corresponding to different conserved regions of eEF-Tu. We sequenced the 5' end of one genomic clone and determined the length of the cloned fragment that was protected by eEF-Tu mRNA in S1 nuclease protection assays. A quantitative S1 nuclease protection assay was used to compare the relative steady-state levels of eEF-Tu mRNA in total mRNA in total RNA isolated from hexamethylene-bisacetamide-induced murine erythroleukemia cells. The results show a dramatic reduction in the steady-state level of eEF-Tu mRNA as differentiation proceeds. A similar reduction in transcription of eEF-Tu mRNA was observed in isolated nuclei. Finally, we examined the in vivo synthesis of eEF-Tu during differentiation and found that it declined in a manner parallel to the decline in the steady-state level of eEF-Tu mRNA. In addition, we have isolated and sequenced a cDNA clone for mouse eEF-Tu. The derived amino acid sequence is compared with sequences from other eucaryotes.
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PMID:Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation. 348 Oct 36

The tRNA-like structure of turnip yellow mosaic virus is known to be efficiently recognized and aminoacylated by valyl-tRNA synthetase. The present work reports domains in the isolated tRNA-like fragment (159 terminal nucleotides at the 3'-end of the two viral RNAs) in contact with purified yeast valyl-tRNA synthetase. These domains were determined in protection experiments using chemical and enzymatic structural probes. In addition, new data, re-enforcing the validity of the tertiary folding model for the native RNA, are given. In particular, at the level of the amino acid accepting arm it was found that the two phosphate groups flanking the three guanine residues of loop I are inaccessible to ethylnitrosourea. This is in agreement with a higher-order structure of this loop involving "pseudo knotting", as proposed by Rietveld et al. (1982). Valyl-tRNA synthetase efficiently protects the viral RNA against digestion by single-strand-specific S1 nuclease at the level of the anticodon loop. With cobra venom ribonuclease, specific for double-stranded regions of RNA, protection was detected on both sides of the anticodon arm and at the 5'-ends of loop I, a region that is involved in the building up of the acceptor arm. Loop II, which is topologically homologous to the T-loop of canonical tRNA was likewise protected. Weak protection was observed between arms I and II, and at the 3'-side of arm V. This arm, located at the 5'-side of arm IV (homologous to the D-arm of tRNA), does not participate in the pseudo-knotted model of the valine acceptor arm. Ethylnitrosourea was used to determine the phosphates of the tRNA-like structure in close contact with the synthetase. These are grouped in several stretches scattered over the RNA molecule. In agreement with the nuclease digestion results, protected phosphates are located in arms I, II, and III. Additionally, this chemical probe permits detection of other protected phosphates on the 3'-side of arm IV and on both sides of arm V. When displayed in the three-dimensional model of the tRNA-like structure, protected areas are localized on both limbs of the L-shaped RNA. It appears that valyl-tRNA synthetase embraces the entire tRNA-like structure. This is reminiscent of the interaction model of canonical yeast tRNAVal with its cognate synthetase.
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PMID:Contact areas of the turnip yellow mosaic virus tRNA-like structure interacting with yeast valyl-tRNA synthetase. 354 Mar 11

We have generated mutants of Escherichia coli formylmethionine initiator tRNA in which one, two, and all three G X C base pairs in the GGGCCC sequence in the anticodon stem are changed to those found in E. coli elongator methionine tRNA. Overproduction of the mutant tRNAs using M13 recombinants as an expression vector and development of a one-step purification scheme allowed us to purify, characterize, and analyze the function of the mutant tRNAs. After aminoacylation and formylation, the function of mutant formylmethionyl tRNAs was analyzed in an MS2 RNA-directed in vitro protein-synthesizing system, in AUG-dependent ribosomal P site binding, and in initiation factor binding. The mutant tRNAs show progressive loss of activity in initiation, the mutant with all three G X C base pairs substituted being the least active. The mutations affect binding to the ribosomal P site. None of the mutations affects binding to initiation factor 2. We also show that there is a progressive increase in accessibility of phosphodiester bonds in the anticodon loop of the three mutants to S1 nuclease, such that the cleavage pattern of the mutant with all three G X C base-pair changes resembles that of elongator tRNAs. These results are consistent with the notion that the contiguous G X C base pairs in the anticodon stem of initiator tRNAs impart on the anticodon loop a unique conformation, which may be important in targeting the initiator tRNA to the ribosomal P site during initiation of protein synthesis.
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PMID:Escherichia coli formylmethionine tRNA: mutations in GGGCCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop. 354 Sep 60

The major transcriptional control sequences of vertebrate mitochondrial DNA lie within the displacement loop region. Transcription events initiating in the displacement loop sequence of the mouse genome were identified by 5' end mapping of primary transcripts by S1 nuclease protection and primer extension techniques. Light-strand transcription initiates at a single site, 165 nucleotides upstream of the major heavy-strand origin of replication. Transcription of the heavy strand occurs at two distinct sites, 5 and 13 nucleotides upstream of the gene for phenylalanyl-tRNA, the first heavy-strand-encoded gene. This spatial relationship of the two transcriptional start sites with each other and with the origin of heavy-strand replication and the gene for tRNAPhe is quite similar to that for human mitochondrial DNA. The predominant form of primary heavy-strand transcript in mouse is a short, ca. 75-nucleotide, RNA containing the sequences of tRNAPhe and a few additional nucleotides at the 5' end of tRNAPhe, suggesting that the processing of tRNA involves independent cleavages at the 5' and 3' ends of tRNA sequences.
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PMID:Identification of primary transcriptional start sites of mouse mitochondrial DNA: accurate in vitro initiation of both heavy- and light-strand transcripts. 378 71


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