Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Under conditions unfavorable to growth, the nematode Caenorhabditis elegans enters a developmentally arrested stage, the dauer larva. We have examined gene expression in the dauer larva and during recovery from the dauer stage. Run-on transcription assays with isolated nuclei reveal a depression of general RNA polymerase II transcription to 11-17% of that in other stages. Transcription of individual gene families (including actin, collagen, hsp70, and histone) is similarly depressed relative to actively growing stages. Dauer larvae are, however, capable of being induced for heat shock messages, indicating that they are competent to initiate and elongate transcripts. For most genes surveyed, reduced transcription in dauer larvae correlates with a decrease in message abundance. Hsp70 mRNA, however, is transcribed at lower rates but accumulates at levels comparable to those in other stages. Interestingly, dauer larvae are 15-fold enriched in a mRNA for a C. elegans hsp90 gene. Hsp90 mRNA accumulation is regulated at least in part by differential stability. Dauer larvae thus appear to have a unique pattern of gene expression. Upon placement in food, dauer larvae reenter the developmental pathway as late-stage larvae. Dauer recovery is accompanied by a temporally regulated sequence of gene expression. At least four distinct patterns of gene expression can be distinguished during exit from the dauer stage. Steady-state levels of hsp70 and polyubiquitin mRNA rise sharply within 75 min of recovery before declining by the fourth hour. Actin and histone mRNAs increase steadily following 2-4 hr of recovery, whereas myosin mRNA increases after 10 hr. In contrast, hsp90 mRNA declines sharply within the first 75 min of recovery. Changes in mRNA populations during dauer formation and exit may be physiologically relevant.
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PMID:Gene expression in the Caenorhabditis elegans dauer larva: developmental regulation of Hsp90 and other genes. 157 99

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca2(+)-calmodulin and binding to actin, actin-tropomyosin, Ca2(+)-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca2(+)-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.
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PMID:The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli. 222 89

Two genomic fragments were isolated from a normal and a dystrophic library containing the 3'OH terminus of the fast isoform of myosin heavy chain gene. Restriction map analysis confirmed that the genes were similar. The sequences coding for myosin were defined and shown to be the same in each genomic fragment. However, using a cDNA clone for tcRNA102 and two specific oligomers for tcRNA102 sequences, we determined that only the genomic fragment from normal chick contained homologous sequences to tcRNA102. Dystrophic chick DNA did not contain these regions of homology. In addition, the normal genomic fragment transcribes tcRNA102 in vitro via RNA polymerase III while the corresponding fragment of DNA from dystrophic chick was inactive. These results suggest that there are detectable differences between the normal and dystrophic genomes in this regard.
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PMID:The localization of a tcRNA102 gene near the 3' OH terminus of a fast myosin heavy chain gene. A comparison between normal and dystrophic chickens. 242 21

One or more of the five acidic amino-terminal residues of skeletal muscle actin have been implicated as being important in a number of actin-related processes. We have constructed a series of actins containing mutations at Asp3 and Asp11 and tested these mutant proteins for their ability to bind to DNase I-agarose, polymerize with rabbit skeletal muscle actin, undergo amino-terminal processing, and bind to the myosin-S1 subfragment. The mutant actins were expressed in vitro using a coupled transcription/translation system which involves the synthesis of mutant RNAs with SP6 RNA polymerase followed by their translation in a rabbit reticulocyte lysate. When Asp3 was changed to Ala, His, or Asn there was no difference in the tested properties as compared to wild type actin. These results suggest that an acidic residue at position 3 is not critical for the actin functions measured. When Asp11 was changed to Glu, Asn, or His or if the conserved Asp-Asn sequence at positions 11 and 12 was reversed, the mutants were able to copolymerize with rabbit skeletal muscle actin and be cross-linked to myosin-S1 to nearly the same extent as wild type actin. However, the amount of in vitro-synthesized actin capable of binding to DNase I-agarose with high affinity or undergoing amino-terminal processing was reduced significantly relative to the wild type actin synthesized in vitro. The Asp11 mutants ran anomalously on native polyacrylamide gels suggestive of a conformational change induced in the actin. Together, these results suggest that Asp11 may be important in proper actin folding and function.
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PMID:Studies on the role of actin's aspartic acid 3 and aspartic acid 11 using oligodeoxynucleotide-directed site-specific mutagenesis. 319 44

Mechanical overload in the heart induces two different types of adaptational mechanisms. (a) From a qualitative point of view, the maximum speed of shortening is depressed in relation to a myosin isoenzymic change responsible for decreased ATPase and, although the relaxation appears normal from a physiological point of view, the existence of an abnormality in Ca2+ uptake in the sarcoplasmic reticulum has been well documented. Both of these processes appear to improve efficiency by decreasing the heat produced per gram of tension. The existence of a large broadening of the action potential has now been well established, but it remains unexplained at the biochemical level. The functioning of mitochondria is rather controversial, and although it has been shown that they are both more abundant and smaller, the reason why their respiratory index changes remains unknown. (b) From a quantitative point of view, the adult heart adapts to overload by increasing its mass. This is mainly a consequence of a hypertrophy of the myocytes and a mitotic multiplication of nonmuscular cells. Data suggest that myocyte amitotic divisions may occur, at least in humans, and perhaps in very sizeable experimental hypertrophy. To this phenomenon has been added the development of polyploidy of myocyte nuclei, which seems to be specific to certain species. The stimulation of protein synthesis occurs very soon after pressure overload, and is delayed in volume overload; protein lysis also increases, although this is controversial. The process occurs whatever the proteins. This is accompanied by increased nuclear activity and a stimulation in RNA synthesis, which is especially precocious for messenger RNA. Among the very early events which could be potential signals for protein synthesis, attention has been focused on polyamine, RNA polymerase, and uridine kinase. The trigger mechanism, of course remains hypothetical. As a trigger for protein synthesis, several data suggest an increase in wall stress and stretch; a drop in efficiency is suggested as a trigger for qualitative changes.
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PMID:Biology of cardiac overload. 621 32

The synthesis and characterization of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) using chemical and enzymatic methods are described. GTP alpha S A (SP diastereomer) can be prepared enzymatically from a chemically synthesized mixture of the diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDP alpha S) with phosphoglycerate kinase. GTP alpha S B (RP diastereomer) can be similarly synthesized with succinyl-CoA synthetase and by back-digesting the small amounts of GTP alpha S A formed with phosphoglycerate kinase. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) serves as the precursor for both GTP beta S A (SP diastereomer), prepared with pyruvate kinase and by back-digesting with glycerol kinase, and GTP beta S B (RP diastereomer), obtained with acetate kinase and by back-digesting with myosin. These analogues can be gamma-32P labeled by 32Pi exchange with either phosphoglycerate kinase-phosphoglyceraldehyde dehydrogenase or succinyl-CoA synthetase. Finally, the interaction of these four nucleotides with acetate kinase, RNA polymerase, and succinyl-CoA synthetase is described.
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PMID:Synthesis and characterization of diastereomers of guanosine 5'-O-(1-thiotriphosphate) and guanosine 5'-O-(2-thiotriphosphate). 709 23

The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.
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PMID:Transcription against an applied force. 750 62

Titin (connectin) is a giant protein that forms a single-molecule elastic filament extending from the M-line to the Z-line in the striated muscle sarcomere. The sequence of titin consists mainly of repeats of two types of approximately 100-amino acid motifs (class I and class II that show homology to the fibronectin type III and immunoglobulin-C2 domains, respectively). To investigate the functions of the two classes of titin motifs as the basic units of this large sarcomere organizer molecule, titin cDNA segments encoding single class I or class II or linked class I-II motifs were cloned by polymerase chain reaction from a rat cardiac cDNA library into the T7 RNA polymerase-based pAED4 vector to express non-fusion titin fragments in Escherichia coli. High level expression of the three titin fragments was achieved, and effective rapid purification procedures were developed. The purified titin fragments were verified by their amino acid composition, apparent molecular mass, and charge. Antibodies raised against the genetically expressed titin motifs specifically recognized intact rat cardiac and skeletal muscle titins in Western blotting and immunofluorescence microscopy, confirming the authenticity of the cloned fragments. High beta-sheet contents of these titin motifs indicate a folding state very similar to that of intact native titin. Solid-phase protein-binding assays demonstrated that a single class I motif was able to bind both myosin and F-actin. In comparison, a single class II motif had weaker binding to only F-actin but the fragment containing linked class I and class II motifs showed significantly stronger interactions with both myosin and F-actin. The binding of titin motifs to myosin supports the proposed association of A-band titin with the thick filament, and the novel titin-F-actin interaction was confirmed by F-actin cosedimentation assays, suggesting that titin may also be involved in the structure and/or function of the thin filament.
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PMID:Cloned rat cardiac titin class I and class II motifs. Expression, purification, characterization, and interaction with F-actin. 789 40

The sigma D form of RNA polymerase from Bacillus subtilis has been shown previously to direct the synthesis of several transcription units bearing genes for flagellin, motility proteins, and autolysins. In this report, we describe an operon of genes transcribed from the sigma D-dependent promoter PD-1. We have identified three complete open reading frames and one partial one downstream of this promoter; immediately upstream is the previously identified comF locus. The PD-1 operon encodes the presumptive B. subtilis homologs of two Salmonella typhimurium late flagellar genes, flgM and flgK. Also present in this operon are two genes of unknown function, orf139 and orf160, whose products show similarities to the eukaryotic cytoskeletal proteins myosin and vimentin, respectively. orf139 and orf160 may encode proteins that form extended alpha-helical secondary structures and coiled-coil quaternary structures which may be filamentous components of the gram-positive bacterial flagellum. We have characterized the B. subtilis flgM gene further by constructing an in-frame deletion mutation, flgM delta 80, and creating strains of B. subtilis in which this allele has replaced the wild-type copy. By primer extension analysis of cellular RNA, we have shown that the flgM delta 80 mutation relieves the block to transcription of two other sigma D-dependent operons imposed by an unlinked mutation in a gene directing early flagellar synthesis. We conclude that, as in the case of S. typhimurium, early flagellar synthesis in B. subtilis is coupled to late flagellar synthesis through repression of sigma D-dependent transcription by the flgM gene product.
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PMID:Identification of flagellar synthesis regulatory and structural genes in a sigma D-dependent operon of Bacillus subtilis. 804 79

rRNA synthesis decreases significantly during the differentiation of rat L6 myoblasts to myotubes. Nuclear run-on assays demonstrated that the decrease was attributable to decreased rates of rRNA gene transcription. Immunoblot analysis indicated a marked reduction in amounts of the RNA polymerase I transcription factors UBF1 and UBF2 (upstream binding factors 1 and 2, respectively). The levels of these factors dropped in parallel with the down-shift in rRNA gene transcription. The amount of UBF does not fall due to a general decrease in cellular protein, as myosin heavy-chain protein accumulates markedly during this same time. RNA blots of total RNA isolated from myoblasts and differentiating myotubes showed a decrease in the mRNA for UBF, at the same time the mRNA for myogenin was accumulating. The down-shift in UBF mRNA levels preceded the decrease in the protein levels for UBF. There have been reports that the acute response of the rRNA gene transcription system to physiological signals in many systems involves an RNA polymerase I-associated factor. However, our results imply that the regulation of rRNA gene DNA transcription in response to physiological processes, such as differentiation, may involve multiple regulatory pathways.
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PMID:Coordinated decreases in rRNA gene transcription factors and rRNA synthesis during muscle cell differentiation. 839 56


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