scholarly journals A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo.

1992 ◽  
Vol 12 (9) ◽  
pp. 4015-4025 ◽  
Author(s):  
R H Morse ◽  
S Y Roth ◽  
R T Simpson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Nucleosome Positioning ◽  
Yeast Cells ◽  
Trna Genes ◽  
Start Site ◽  
Cis Acting

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.

A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo

1992 ◽  
Vol 12 (9) ◽  
pp. 4015-4025
Author(s):  
R H Morse ◽  
S Y Roth ◽  
R T Simpson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Nucleosome Positioning ◽  
Yeast Cells ◽  
Trna Genes ◽  
Start Site ◽  
Cis Acting

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.

scholarly journals Template Structural Requirements for Transcription In Vivo by RNA Polymerase II

1982 ◽  
Vol 2 (12) ◽  
pp. 1595-1607 ◽  
Author(s):  
Timothy J. Miller ◽  
Janet E. Mertz
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Structural Requirements ◽  
Infected Monkey ◽  
Full Complement ◽  
Sv40 Dna

Purified simian virus 40 (SV40) DNA is reconstituted into chromatin and transcribed by endogenous RNA polymerase II when microinjected into nuclei ofXenopus laevisoocytes. We have correlated the kinetics of chromatin reconstitution with that of accumulation of virus-specific RNA in this system. A delay of approximately 3 h was found in the appearance of appreciable numbers of both fully supercoiled molecules and transcriptionally active templates. SV40 minichromosomes, isolated from virus-infected monkey cells with 0.2 M NaCl, also exhibited this lag in onset of transcriptional activity when microinjected into oocytes. These findings indicate that neither purified SV40 DNA nor SV40 DNA containing a full complement of nucleosomes can function as a template for transcription in vivo before association with appropriate cellular nonhistone chromosomal factors has taken place. In addition, the gradual degradation of linear SV40 DNA in oocytes was not sufficient to account for the fact that it was much less transcriptionally active than circular SV40 DNA. Taken together, these results indicate that the conformational state of the DNA can affect its ability to function as a template for transcription in vivo by RNA polymerase II. In contrast, transcription by RNA polymerase III of purified, circularized cloned DNAs encoding genes for 5S rRNA was detectable long before the injected DNAs had time to reconstitute into chromatin. Therefore, the template structural requirements for transcription in vivo by RNA polymerases II and III are different.

scholarly journals Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.

1992 ◽  
Vol 12 (10) ◽  
pp. 4271-4278 ◽  
Author(s):  
D E Syroid ◽  
R I Tapping ◽  
J P Capone
Keyword(s):  
Mammalian Cells ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Lac Repressor ◽  
Trna Genes ◽  
Coding Region ◽  
Lac Operator ◽  
Cell Extracts

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

scholarly journals Promoter-Specific Shifts in Transcription Initiation Conferred by Yeast TFIIB Mutations Are Determined by the Sequence in the Immediate Vicinity of the Start Sites

2001 ◽  
Vol 21 (14) ◽  
pp. 4427-4440 ◽  
Author(s):  
Silviu L. Faitar ◽  
Seth A. Brodie ◽  
Alfred S. Ponticelli
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Genetic Selection ◽  
Functional Domain ◽  
Start Site ◽  
Recessive Mutations ◽  
Transcription Factor Iib ◽  
General Transcription Factor

ABSTRACT The general transcription factor IIB (TFIIB) is required for transcription of class II genes by RNA polymerase II. Previous studies demonstrated that mutations in the Saccharomyces cerevisiae SUA7 gene, which encodes TFIIB, can alter transcription initiation patterns in vivo. To further delineate the functional domain and residues of TFIIB involved in transcription start site utilization, a genetic selection was used to isolate S. cerevisiae TFIIB mutants exhibiting downstream shifts in transcription initiation in vivo. Both dominant and recessive mutations conferring downstream shifts were identified at multiple positions within a highly conserved homology block in the N-terminal region of the protein. The TFIIB mutations conferred downstream shifts in transcription initiation at the ADH1 and CYC1 promoters, whereas no significant shifts were observed at the HIS3 promoter. Analysis of a series of ADH1-HIS3 hybrid promoters and variant ADH1 and HIS3 promoters containing insertions, deletions, or site-directed base substitutions revealed that the feature that renders a promoter sensitive to TFIIB mutations is the sequence in the immediate vicinity of the normal start sites. We discuss these results in light of possible models for the mechanism of start site utilization by S. cerevisiae RNA polymerase II and the role played by TFIIB.

scholarly journals Purines are required at the 5' ends of newly initiated RNAs for optimal RNA polymerase III gene expression.

1996 ◽  
Vol 16 (10) ◽  
pp. 5801-5810 ◽  
Author(s):  
G N Zecherle ◽  
S Whelen ◽  
B D Hall
Keyword(s):  
Rna Polymerase ◽  
Ternary Complex ◽  
Rna Polymerase Iii ◽  
Phenotypic Change ◽  
Start Site ◽  
Transcription Start ◽  
Polymerase Iii ◽  
Nascent Transcripts ◽  
Mutant Genes

We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene. The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1. Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding the +1 and +2 nucleotides of pre-tRNA Tyr, had a strong deleterious effect in vivo, as did the more extensive change CTCCTC. In contrast, mutant genes bearing each of the possible single changes at nucleotide +1 retained normal suppression levels. The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site. ATP was preferentially utilized as the 5' nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the +1 nucleotide. Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex. Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o. Our results suggest that the purine-rich sequences at the 5' ends of the nascent transcripts of many genes act to stabilize the early ternary complex.

scholarly journals Induced RPB1 depletion reveals a direct gene-specific control of RNA Polymerase III function by RNA Polymerase II

2019 ◽  
Author(s):  
Alan Gerber ◽  
Keiichi Ito ◽  
Chi-Shuen Chu ◽  
Robert G. Roeder
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Serum Starvation ◽  
Trna Genes ◽  
Large Set ◽  
Specific Expression ◽  
Pol Ii ◽  
Direct Gene ◽  
Specific Regulation

SummaryIncreasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.

Expression and function of a human initiator tRNA gene in the yeast Saccharomyces cerevisiae

1990 ◽  
Vol 10 (9) ◽  
pp. 4486-4494
Author(s):  
M A Francis ◽  
U L Rajbhandary
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Functional Expression ◽  
Yeast Cells ◽  
Trna Genes ◽  
Flanking Sequence ◽  
Coding Sequence ◽  
Initiator Trna

We showed previously that the human initiator tRNA gene, in the context of its own 5'- and 3'-flanking sequences, was not expressed in Saccharomyces cerevisiae. Here we show that switching its 5'-flanking sequence with that of a yeast arginine tRNA gene allows its functional expression in yeast cells. The human initiator tRNA coding sequence was either cloned downstream of the yeast arginine tRNA gene, with various lengths of intergenic spacer separating them, or linked directly to the 5'-flanking sequence of the yeast arginine tRNA coding sequence. The human initiator tRNA made in yeast cells can be aminoacylated with methionine, and it was clearly separated from the yeast initiator and elongator methionine tRNAs by RPC-5 column chromatography. It was also functional in yeast cells. Expression of the human initiator tRNA in transformants of a slow-growing mutant yeast strain, in which three of the four endogenous initiator tRNA genes had been inactivated by gene disruption, resulted in enhancement of the growth rate. The degree of growth rate enhancement correlated with the steady-state levels of human tRNA in the transformants. Besides providing a possible assay for in vivo function of mutant human initiator tRNAs, this work represents the only example of the functional expression of a vertebrate RNA polymerase III-transcribed gene in yeast cells.

Gene insulation. Part I: natural strategies in yeast and Drosophila

2010 ◽  
Vol 88 (6) ◽  
pp. 875-884 ◽  
Author(s):  
Michèle Amouyal
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Eukaryotic Cells ◽  
Trna Genes ◽  
Initiation Complex ◽  
Heterochromatin Formation ◽  
Transcription Initiation Complex

This review in two parts deals with the increasing number of processes known to be used by eukaryotic cells to protect gene expression from undesired genomic enhancer or chromatin effects, by means of the so-called insulators or barriers. The most advanced studies in this expanding field concern yeasts and Drosophila (this article) and the vertebrates (next article in this issue). Clearly, the cell makes use of every gene context to find the appropriate, economic, solution. Thus, besides the elements formerly identified and specifically dedicated to insulation, a number of unexpected elements are diverted from their usual function to structure the genome and enhancer action or to prevent heterochromatin spreading. They are, for instance, genes actively transcribed by RNA polymerase II or III, partial elements of these transcriptional machineries (stalled RNA polymerase II, normally required by genes that must respond quickly to stimuli, or TFIIIC bound at its B-box, normally required by RNA polymerase III for assembly of the transcription initiation complex at tRNA genes), or genomic sequences occupied by variants of standard histones, which, being rapidly and permanently replaced, impede heterochromatin formation.

scholarly journals Identifying a species-specific region of yeast TF11B in vivo.

1996 ◽  
Vol 16 (7) ◽  
pp. 3651-3657 ◽  
Author(s):  
S P Shaw ◽  
J Wingfield ◽  
M J Dorsey ◽  
J Ma
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Specific Region ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Species Specific

The general transcription factor IIB (TFIIB) is required for RNA polymerase II transcription in eukaryotes. It provides a physical link between the TATA-binding protein (TBP) and the RNA polymerase and is a component previously suggested to respond to transcriptional activators in vitro. In this report, we compare the yeast (Saccharomyces cerevisiae) and human forms of the protein in yeast cells to study their functional differences. We demonstrate that human TFIIB fails to functionally replace yeast TFIIB in yeast cells. By analyzing various human-yeast hybrid TFIIB molecules, we show that a 14-amino-acid region at the amino terminus of the first repeat of yeast TFIIB plays an important role in determining species specificity in vivo. In addition, we identify four amino acids in this region that are critical for an amphipathic helix unique to yeast TFIIB. By site-directed mutagenesis analyses we demonstrate that these four amino acids are important for yeast TFIIB's activity in vivo. Finally, we show that mutations in the species-specific region of yeast TFIIB can differentially affect the expression of genes activated by different activators in vivo. These results provide strong evidence suggesting that yeast TFIIB is involved in the process of transcriptional activation in living cells.

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