Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

To investigate the identity determinants of E. coli threonine tRNA, various transcripts were prepared by in vitro transcription system with T7 RNA polymerase. Substitutions of the anticodon second letter G35 and the third letter U36 to other nucleotides led to a remarkable decrease of threonine charging activity. Charging experiments with a series of anticodon-deletion transcripts also suggest the importance of the G35U36 sequence. A mutation at either the G1-C72 or C2-G71 base pair in the acceptor stem seriously affected the threonine charging activity. These results indicate that the second and third positions of the anticodon and the first and second base pairs in the acceptor stem are the recognition sites of E. coli tRNA(THR) for threonyl-tRNA synthetase. Discriminator base, A73, is not involved in threonine charging activity.
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PMID:Identity determinants of E. coli threonine tRNA. 156 50

Using temperature- and proteolytically sensitive derivatives to inactivate the function of the yeast TATA-binding protein (TBP) in vivo, we investigated the requirement of TBP for transcription by the three nuclear RNA polymerases in yeast cells. TBP is required for RNA polymerase II (pol II) transcription from promoters containing conventional TATA elements as well as functionally distinct promoters that lack TATA-like sequences. TBP is also required for transcription of the U6 snRNA and two different tRNA genes mediated by RNA pol III as well as transcription of ribosomal RNA mediated by RNA pol I. For all promoters tested, transcription decreases rapidly and specifically upon inactivation of TBP, strongly suggesting that TBP is directly involved in the transcription process. These observations suggest that TBP is required for transcription of all nuclearly encoded genes in yeast, although distinct molecular mechanisms are probably involved for the three RNA polymerase transcription machineries.
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PMID:The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells. 158 47

Apparent Michaelis constants for nucleotides in transcription of yeast tRNA gene by homologous RNA polymerase III with auxiliary protein factors, were found to be remarkably higher in initiation than in elongation of RNA chain. This supports presumptions regarding topological similarities between catalytic centers of bacterial and eukaryotic RNA polymerases.
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PMID:Relative affinities of nucleotide substrates for the yeast tRNA gene transcription complex. 159 Aug 92

Transcription of Xenopus laevis U1 snRNA genes is subject to a precise program with respect both to the timing of activation at the midblastula transition (MBT) and to the relative levels of the two embryonic U1 RNAs (xU1b1 and b2) that are made. Here, we demonstrate that exogenous xU1b genes injected into developing X. laevis embryos come under the same controls as the endogenous genes. Injected U1 genes, unlike exogenous RNA polymerase III genes, remain quiescent until MBT and their activation at MBT requires protein synthesis during the early cleavage stages. Significantly, the onset of 4-8S RNA transcription occurs at the normal time, even when the DNA content of the embryo has been increased by injection of exogenous DNA or reduced through cleavage arrest, indicating that transcriptional activation at MBT is independent of the ratio of DNA (nucleus) to cytoplasm. In cleavage-arrested (coenocytic) embryos, the reduced level of DNA at MBT results both in a decrease in snRNA and tRNA synthesis (reflecting the lower gene dosage) and in a prolonged synthesis of large amounts of unusual RNA polymerase III transcripts, OAX RNAs. In normally cleaving embryos, small amounts of these unstable OAX RNAs (encoded by satellite I DNA) are synthesized only briefly at MBT. Our demonstration that RNA and DNA metabolism is aberrant in cleavage-arrested embryos requires reevaluation of previous experiments on transcriptional activation that utilized such coenocytic embryos.
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PMID:Control of 4-8S RNA transcription at the midblastula transition in Xenopus laevis embryos. 159 58

Albino rice plants derived from pollen contain plastid genomes that have suffered large-scale deletions. From the roots of albino plants, we obtained several calli containing homogeneous plastid DNA differing in the size and position of the deletion. DNA differing in the size and position of the deletion. Southern blotting and pulsed field gel electrophoresis experiments revealed that the DNAs were linear molecules having a hairpin structure at both termini, existing as monomers (19 kb) or dimers, trimers and tetramers linked to form head-to-head and tail-to-tail multimers. This characteristic form is similar to that of the vaccinia virus, in which the replication origin is thought to lie at or near the hairpin termini. Furthermore, polymerase chain reaction experiments revealed complete loss of the ribosomal RNA genes of the plastid DNA. The results suggest that plant cells can grow without translation occurring in plastids. All of the deleted plastid DNAs commonly retained the region containing the tRNA(Glu) gene (trnE), which is essential for biosynthesis of porphyrin. As porphyrin is the precursor of heme for mitochondria and other organelles, it is considered that trnE on the remnant plastid genome may be transcribed by an RNA polymerase encoded on nuclear DNA.
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PMID:Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth. 160 57

We have recently identified a tRNA gene cluster in the Arabidopsis nuclear genome. One tRNA(Ser) (AGA) gene and two tRNA(Tyr) (GTA) genes occur in tandem arrangement on a 1.5 kb unit that is amplified about 20-fold at a single chromosomal site. Here we have studied the in vitro expression of seven individually cloned tRNA(Ser) genes (pAtS1 to pAtS7) derived from this cluster. Five out of the seven tRNA(Ser) genes contain point mutations in the coding region which have in part adverse effects on the expression of these genes in different cell-free systems: (i) C10 and A62 in plant tRNA(Ser) genes, which correspond to G10 and C62, respectively, in all known vertebrate tRNA genes, result in a reduced transcription efficiency in HeLa but not in yeast extract. This indicates that yeast RNA polymerase III tolerates nucleotide substitutions at positions 10 [5' internal control region (ICR)] and 62 (3' ICR), whereas the vertebrate RNA polymerase III requires a more stringent consensus sequence. (ii) Processing of a pre-tRNA(Ser) with a mismatch in the aminoacyl stem is impaired in HeLa, yeast and wheat germ extracts, however, a mismatch in the anticodon stem is deleterious for HeLa and wheat germ but not for yeast processing enzymes. The unexpectedly high number of potential tRNA(Ser) pseudogenes in the cluster - quite in contrast to the tRNA(Ser) genes which mainly code for functional tRNAs - suggested that tRNA(Ser) (AGA) genes also occur elsewhere in the genome. We present evidence that single copies of tRNA(Ser) (AGA) genes do indeed exist outside the tRNA gene cluster.
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PMID:Expression of variant nuclear Arabidopsis tRNA(Ser) genes and pre-tRNA maturation differ in HeLa, yeast and wheat germ extracts. 160 62

The Saccharomyces cerevisiae transcription factors (TF) IIIB and IIIC assemble onto their respective DNA-binding sites on the SUP4 tRNA(Tyr) gene at 0 degrees C. RNA polymerase III specifically associates at 0 degrees C with this TFIIIC-TFIIIB-DNA complex to form a stable "closed" promoter complex in which the DNA surrounding the transcriptional start retains its duplex form. Promoter "opening" is a temperature-dependent and readily reversible process that involves up to 22 unwound base-pairs of DNA, and can be followed by analyzing the hyperreactivity of thymine to KMnO4 oxidation. This promoter opening increases progressively from 10 degrees C to 40 degrees C, with at least two regions within the transcription bubble appearing to melt independently. In contrast, the temperature dependence of forming an initiated transcription complex containing a 17 nucleotide nascent RNA chain displays a sharp transition between 10 degrees C and 15 degrees C. When RNA polymerase initiates transcription under conditions that limit the nascent RNA chain to less than six nucleotides, there is no displacement of the transcription bubble. These transcription complexes are distinguishable from "open" promoter complexes in their maintenance of the transcription bubble at 0 degrees C, and from transcription complexes with more extended (17 nucleotide) RNA chains in their sensitivity to disruption by heparin. In light of recent results by others that demonstrate a requirement for an RNA transcription factor in a Bombyx mori-based in vitro RNA polymerase III transcription system, we have searched for a comparable component in the S. cerevisiae-derived system. We show that if an RNA component is required in the yeast-derived system, it is not susceptible to inactivation by massive amounts of micrococcal nuclease, RNase A, or RNase T1.
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PMID:Formation of open and elongating transcription complexes by RNA polymerase III. 161 62

Inverted sequences of the chloramphenicol acetyltransferase (CAT) reporter gene were fused to a soybean tRNA(met(i)) gene lacking a terminator such that the tRNA(met(i)) sequences caused the co-transcription of CAT antisense sequences by RNA polymerase III. When electroporated into carrot protoplasts, these antisense DNA constructs suppressed CAT enzyme activity expressed from co-electroporated DNAs containing the CAT gene downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. Our most effective construct, an antisense sequence complementary to the 3' portion of the CAT gene, inhibited CAT activity five-fold greater than an antisense construct expressed by RNA polymerase II from the cauliflower mosaic virus 35S RNA promoter. These results indicate that antisense sequences transcribed by RNA polymerase III should efficiently suppress gene expression in plants.
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PMID:Suppression of gene expression in plant cells utilizing antisense sequences transcribed by RNA polymerase III. 162 77

A coupled translation-transcription cell-free system was established from eukaryotic cells. The biosynthetic activity of this coupled system closely resembles the synthetic behavior of cells in vivo, and exhibits regulatory phenomena similar to that of intact cells. The translational system consists of rabbit reticulocyte lysate, or its components fractionated by centrifugation. The transcriptional portion consists of cockerel liver nuclei. Incorporation of amino acids into protein by the coupled system is linear for hours. Similarly, transcription in the coupled system is continuous for hours and is proportional with time. More than 90% of the transcriptional products are secreted into the incubation medium. The components of the translational system influence and regulate transcriptional activities. In the presence of ribosomes the nuclei transcribe mostly poly(A)+ RNA with alpha-amanitin sensitivity consistent with activation of RNA polymerase II. Hybrid selection experiments demonstrate authentic preproalbumin mRNA among the transcriptional products. The putative mRNA secreted into the medium in the coupled system is found on polysomes, indicating translation of de novo synthesized message. Addition of excess reticulocyte mRNP to the medium of the coupled system results in transcription of primarily ribosomal RNA, 5S RNA, and tRNA, the products of RNA polymerases I and III. These activities closely imitate the behavior of liver in vivo under conditions of nutritional shifts or hormonal influences. The coupled system transcribes, processes, and transports substantial quantities of RNA, about 1.6 micrograms/10(6) nuclei/h. Thus, a coupled system has been established that lends itself to the exploration of regulatory interactions of cell components as it appears to closely resemble the in vivo situation.
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PMID:Regulation of transcription by translational components in coupled translation-transcription cell-free system. 163 75

PCF1-1 is a dominant suppressor of a tRNA gene A block promoter mutation (A19) in Saccharomyces cerevisiae. Transcriptional activation by PCF1-1 was examined in vitro using whole-cell extracts and purified factors derived from mutant and wild-type strains. These experiments show that PCF1 is a general activator of RNA polymerase III (pol III) gene transcription. The transcription of all pol III genes analyzed to date, including type I and numerous type II genes, is increased 3-7 fold in mutant cell extracts. Single round transcription assays indicate that the PCF1-1 mutation increases the number of functional preinitiation complexes and suggest that this is achieved by increasing the intrinsic activity of the encoded product rather than its amount. Point mutations throughout the A block of the sup3-e gene and numerous B block mutations fail to abolish transcriptional activation suggesting that interactions between TFIIIC and the internal promoter are unaffected by PCF1-1. Moreover, TFIIIC purified from the mutant strain is incapable of conferring PCF1-1 transcriptional activity to a reaction in which the remaining components are wild-type. In contrast, the activity of the TFIIIB fraction is increased in PCF1-1 extracts and can reconstitute mutant levels of transcription when added to wild-type TFIIIC and polymerase. We conclude that PCF1 is a component or regulator of TFIIIB.
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PMID:The PCF1-1 mutation increases the activity of the transcription factor (TF) IIIB fraction from Saccharomyces cerevisiae. 164 38


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