Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5' untranslated region; petD encodes subunit IV of the cytochrome b(6)/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5' end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5'-->3' exoribonucleolytic degradation. Elements II and III, located in the center and near the 3' end of the 5' untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.
Mol Cell Biol 1999 Dec
PMID:Small cis-acting sequences that specify secondary structures in a chloroplast mRNA are essential for RNA stability and translation. 1056 73

The contribution of the tRNA "core" to aminoacylation is beginning to be recognized. One example is the core region of Escherichia coli tRNA(Cys), which has been shown by biochemical studies to be important for aminoacylation. This core has several layers of unusual base-pairs, which are revealed by the recent crystal structure of the tRNA complexed with the elongation factor EF-Tu and an analog of GTP. One of these layers consists of a 9:[13:22] base-triple, rather than the 46:[13:22] or 45:[13:22] base-triple that is commonly observed in tRNA structure. Because 13:22 is an important element in aminoacylation of E. coli tRNA(Cys), a better understanding of its structure in the tRNA core will shed light on its role in aminoacylation. In this study, we used the phage T7 transcript of the tRNA as a substrate. We probed the structure of 13:22 by dimethyl sulfate and tested its partner in a base-triple by generating mutations that could be assayed for aminoacylation. The results of this study in general are in a better agreement with a 46:[13:22] base-triple that we previously proposed. Although these results are not interpreted as direct proof for the 46:[13:22] base-triple, they shed new light on features of the tRNA core that are important for aminoacylation.
J Mol Biol 2000 Jan 28
PMID:Probing a tRNA core that contributes to aminoacylation. 1065 90

Telomerase is a reverse transcriptase that adds single-stranded telomeric repeats to the ends of linear eukaryotic chromosomes. It consists of an RNA molecule including a template sequence, a protein subunit containing reverse transcriptase motifs, and auxiliary proteins. We have carried out an interference footprinting analysis of the Tetrahymena telomerase elongation complexes. In this study, single-stranded oligonucleotide primers containing telomeric sequences were modified with base-specific chemical reagents and extended with the telomerase by a single (32)P-labeled dGMP or dTMP. Base modifications that interfered with the primer extension reactions were mapped by footprinting. Major functional interactions were detected between the telomerase and the six or seven 3'-terminal residues of the primers. These interactions occurred not only with the RNA template region, but also with another region in the enzyme ribonucleoprotein complex designated the telomerase DNA interacting surface (TDIS). This was indicated by footprints generated with dimethyl sulfate (that did not affect Watson-Crick hydrogen bonding) and by footprinting assays performed with mutant primers. In primers aligned at a distance of 2 nucleotides along the RNA template region, the footprints of the six or seven 3'-terminal residues were shifted by 2 nucleotides. This shift indicated that during the elongation reaction, TDIS moved in concert with the 3' ends of the primers relative to the template region. Weak interactions occurred between the telomerase and residues located upstream of the seventh nucleotide. These interactions were stronger in primers that were impaired in the ability to align with the template.
Mol Cell Biol 2000 Jun
PMID:Interference footprinting analysis of telomerase elongation complexes. 1082 87

DMSO reductase (DMSOR) from Rhodobacter capsulatus, well-characterised as a molybdoenzyme, will bind tungsten. Protein crystallography has shown that tungsten in W-DMSOR is ligated by the dithiolene group of the two pyranopterins, the oxygen atom of Ser147 plus another oxygen atom, and is located in a very similar site to that of molybdenum in Mo-DMSOR. These conclusions are consistent with W L(III)-edge X-ray absorption, EPR and UV/visible spectroscopic data. W-DMSOR is significantly more active than Mo-DMSOR in catalysing the reduction of DMSO but, in contrast to the latter, shows no significant ability to catalyse the oxidation of DMS.
J Mol Biol 2000 Jun 09
PMID:Dimethylsulfoxide reductase: an enzyme capable of catalysis with either molybdenum or tungsten at the active site. 1083 70

Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.
J Mol Biol 2000 May 26
PMID:Interaction of translation initiation factor IF1 with the E. coli ribosomal A site. 1086 Jul 19

Ectopic secretion of ACTH, from sites such as small cell lung cancer (SCLC), results in severe Cushing's syndrome. ACTH is cleaved from POMC. The syndrome may occur when the highly tissue-specific promoter of the human POMC gene (POMC) is activated. The mechanism of activation is not fully understood. This promoter is embedded within a defined CpG island, and CpG islands are usually considered to be unmethylated in all tissues. We demonstrate that much of this CpG island is methylated in normal nonexpressing tissues, in contrast to somatically expressed CpG island promoters reported to date, and is specifically unmethylated in expressing tissues, tumors, and the POMC-expressing DMS-79 SCLC cell line. A narrow 100-bp region is free of methylation in all tissues. E2F factors binding to the upstream domain IV region of the promoter have been shown to be involved in the expression of POMC in SCLC. We show that these sites are methylated in normal nonexpressing tissues, which will prevent binding of E2F, but are unmethylated in expressing tissue. Methylation in vitro is sufficient for silencing of expression, which is not reversed by treatment with Trichostatin A, suggesting that inhibition of expression may be mediated by means other than recruitment of histone deacetylase activity. The DMS-79 cells lack POMC demethylating activity, implying that the methylation and expression patterns are likely to be set early or before neoplastic transformation, and that targeted de novo methylation might be a potential therapeutic strategy.
Mol Endocrinol 2001 Feb
PMID:The CpG island promoter of the human proopiomelanocortin gene is methylated in nonexpressing normal tissue and tumors and represses expression. 1115 38

ACTH-producing tumors of nonpituitary origin characteristically exhibit insensitivity to the negative feedback effects of glucocorticoids. In the DMS-79 cell line derived from an ACTH-producing small cell lung cancer we have previously identified an aberrantly spliced glucocorticoid receptor (GRDelta) that lacks a ligand-binding domain. We examined the interactions of this truncated form of GR with the proximal human proopiomelanocortin (POMC) promoter. In electrophoretic mobility shift assays GRDelta bound to the negative glucocorticoid response element (nGRE) at position -78 to -50 in the human POMC promoter. Nur77, an orphan nuclear receptor that exerts positive regulatory effects on the POMC gene is also known to bind to this DNA element. The functional properties of GR and GRDelta binding to this DNA element were examined in transient transfection experiments in murine AtT-20 corticotroph tumor cells. Reporter gene expression under the control of proximal POMC promoter elements was stimulated by addition of forskolin to the culture medium or by transfection with expression constructs for human Nak1, the human homologue of Nur77. Treatment of transfected cells with dexamethasone resulted in suppression of forskolin- or Nak1-stimulated POMC-reporter gene expression in the presence of co-transfected GR but not with GRDelta. The experiments indicate that in the human POMC promoter GRDelta is capable of binding to the nGRE but cannot effect trans-repression of POMC-reporter gene expression.
J Mol Endocrinol 2001 Feb
PMID:Function of a truncated glucocorticoid receptor form at a negative glucocorticoid response element in the proopiomelanocortin gene. 1117 53

We determined the adduct maps of S(N)1 and S(N)2 alkylating agents in cultured human cells (in vivo) and in vitro to probe DNA-protein interactions along sequences of the promoter and exon 1 of the Fragile-X mental retardation 1 (FMR1) gene. Using ligation-mediated polymerase chain reaction (LMPCR), we compared the piperidine-sensitive alkylpurines sites generated by treating cultured cells (in vivo) and naked DNA (in vitro) with S(N)1 (N-methyl-N-nitrosourea, N-nitroso(acetoxymethyl)methylamine and 1-methyl-3-nitro-1-nitrosoguanidine) and S(N)2 alkylating agents (dimethyl sulfate (DMS), methane sulfonic acid methyl ester, iodo methane, diethyl sulfate, methane sulfonic acid ethyl ester and iodo ethane). The FMR1 promoter has four sites where DNA-protein interactions are observed. In these regions, the S(N)1 methylating agent reactions produced only hypo-reactive sites. In contrast, iodoalkane S(N)2 alkylating agents (MeI and EtI) reactions generated only hyper-reactive sites. Although there are hyper-reactive sites for the other S(N)2 reagents, the hyper-reactive site at +14 on the FMR1 map is more pronounced for the sulfate and sulfonate-derived alkylating agents than for the iodoalkanes. However, DMS modification in the presence of methyl sulfone, a compound that does not alkylate DNA, eliminates the hyper-reactive site observed at +14. This suggests that the electron-rich oxygen atoms of the sulfate and sulfonate-derived S(N)2 alkylating agent structure position the alkylating moiety to the neighboring N-7-guanine position to favor alkyl transfer to the guanine. Using KMnO(4) to probe for single-strand DNA, an unpaired cytosine base was detected at the 5'-side of the hyper- reactive guanine base at position +14, consistent with the formation of a local DNA single-strand bulge. In conclusion, we show that the sequence context-dependent formation of alkylpurines is determined by the chemical nature of the alkylating agent, the DNA sequence context, chromatin structure, and the presence of other non-reactive molecules that can inhibit alkylation.
J Mol Biol 2001 Feb 16
PMID:Alkylating agent and chromatin structure determine sequence context-dependent formation of alkylpurines. 1123 92

Upon binding of a decamer bis-PNA (H-Lys-TTCCTCTCTT-(eg1)(3)-TTCTCTCCTT-LysNH(2)) to a complementary target in a double-stranded DNA fragment, three distinct complexes were detected by gel mobility shift analysis. Using in situ chemical probing techniques (KMnO(4) and DMS) it was found that all three complexes represent bona fide sequence-specific PNA binding to the designated target, but the complexes were structurally different. One complex that preferentially formed at higher PNA concentrations contains two bis-PNA molecules per DNA target, whereas the other two complexes are genuine triplex invasion clamped structures. However, these two latter complexes differ by the path relative to the DNA target of the flexible ethylene-glycol linker connecting the two PNA oligomers that comprise a bis-PNA. We distinguish between one in which the linker wraps around the non-target DNA strand, thus making this strand part of the triplex invasion complex and another complex that encompass the target strand only. The implications of these results are discussed in terms of DNA targeting by synthetic ligands.
J Mol Biol 2001 Mar 16
PMID:Structural isomers of bis-PNA bound to a target in duplex DNA. 1124 4

Quantitative analysis of multiple-hit potassium permanganate (KMnO(4)) footprinting has been carried out in vivo on Saccharomyces cerevisiae 5S rRNA genes. The results fix the number of open complexes at steady state in exponentially growing cells at between 8 and 17% of the 150 to 200 chromosomal copies. UV and dimethyl sulfate footprinting set the transcription factor TFIIIB occupancy at 23 to 47%. The comparison between the two values suggests that RNA polymerase III binding or promoter opening is the rate-limiting step in 5S rRNA transcription in vivo. Inhibition of RNA elongation in vivo by cordycepin confirms this result. An experimental system that is capable of providing information on the mechanistic steps involved in regulatory events in S. cerevisiae cells has been established.
Mol Cell Biol 2001 May
PMID:RNA polymerase III transcription complexes on chromosomal 5S rRNA genes in vivo: TFIIIB occupancy and promoter opening. 1128 21


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