scholarly journals Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

1994 ◽  
Vol 14 (3) ◽  
pp. 1806-1814 ◽  
Author(s):  
H S Sullivan ◽  
L S Young ◽  
C N White ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Silk Gland ◽  
Flanking Sequence ◽  
Transcription Machinery ◽  
Polymerase Iii ◽  
Active Transcription ◽  
The Difference ◽  
Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions

1994 ◽  
Vol 14 (3) ◽  
pp. 1806-1814
Author(s):  
H S Sullivan ◽  
L S Young ◽  
C N White ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Silk Gland ◽  
Flanking Sequence ◽  
Transcription Machinery ◽  
Polymerase Iii ◽  
Active Transcription ◽  
The Difference ◽  
Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

scholarly journals Induction of Drosophila RNA polymerase III gene expression by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is mediated by transcription factor IIIB.

1994 ◽  
Vol 14 (1) ◽  
pp. 339-347 ◽  
Author(s):  
M E Garber ◽  
A Vilalta ◽  
D L Johnson
Keyword(s):  
Rna Polymerase ◽  
Phorbol Ester ◽  
Rna Polymerase Iii ◽  
Protein Subunit ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Gene Complexes ◽  
Transcription Complexes ◽  
Dna Template

We have previously found that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces specific transcription of tRNA and 5S RNA genes in Drosophila Schneider S-2 cells (M. Garber, S. Panchanathan, R. F. Fan, and D. L. Johnson, J. Biol. Chem. 266:20598-20601, 1991). Having derived cellular extracts from TPA-treated cells, that are capable of reproducing this stimulation in vitro, we have examined the mechanism for this regulatory event. Using conditions that limit reinitiation and produce single rounds of transcription from active gene complexes, we find that the number of functional transcription complexes is increased in extracts prepared from TPA-induced cells. We have analyzed the activities of the transcription factors TFIIIB and TFIIIC derived from extracts prepared from TPA-induced and noninduced cells. Examination of the relative activities of TFIIIC showed that both its ability to reconstitute transcription with TFIIIB and RNA polymerase III and its ability to stably bind to the DNA template are unchanged. However, the activity of TFIIIB derived from the TPA-induced cells is substantially increased compared with that derived from the noninduced cells. The differences in TFIIIB activity account for the differences in the overall transcriptional activities observed in the unfractionated extracts. Western blot analysis of the TATA-binding protein subunit of TFIIIB revealed that there is an increase in the amount of this polypeptide present in the induced cell extracts and TFIIIB fraction. Together, these results indicate that the TPA response in Drosophila cells stimulates specific transcription of RNA polymerase III genes by increasing the activity of the limiting transcription component, TFIIIB, and thereby increasing the number of functional transcription complexes.

Induction of Drosophila RNA polymerase III gene expression by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is mediated by transcription factor IIIB

1994 ◽  
Vol 14 (1) ◽  
pp. 339-347
Author(s):  
M E Garber ◽  
A Vilalta ◽  
D L Johnson
Keyword(s):  
Rna Polymerase ◽  
Phorbol Ester ◽  
Rna Polymerase Iii ◽  
Protein Subunit ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Gene Complexes ◽  
Transcription Complexes ◽  
Dna Template

We have previously found that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces specific transcription of tRNA and 5S RNA genes in Drosophila Schneider S-2 cells (M. Garber, S. Panchanathan, R. F. Fan, and D. L. Johnson, J. Biol. Chem. 266:20598-20601, 1991). Having derived cellular extracts from TPA-treated cells, that are capable of reproducing this stimulation in vitro, we have examined the mechanism for this regulatory event. Using conditions that limit reinitiation and produce single rounds of transcription from active gene complexes, we find that the number of functional transcription complexes is increased in extracts prepared from TPA-induced cells. We have analyzed the activities of the transcription factors TFIIIB and TFIIIC derived from extracts prepared from TPA-induced and noninduced cells. Examination of the relative activities of TFIIIC showed that both its ability to reconstitute transcription with TFIIIB and RNA polymerase III and its ability to stably bind to the DNA template are unchanged. However, the activity of TFIIIB derived from the TPA-induced cells is substantially increased compared with that derived from the noninduced cells. The differences in TFIIIB activity account for the differences in the overall transcriptional activities observed in the unfractionated extracts. Western blot analysis of the TATA-binding protein subunit of TFIIIB revealed that there is an increase in the amount of this polypeptide present in the induced cell extracts and TFIIIB fraction. Together, these results indicate that the TPA response in Drosophila cells stimulates specific transcription of RNA polymerase III genes by increasing the activity of the limiting transcription component, TFIIIB, and thereby increasing the number of functional transcription complexes.

scholarly journals Upstream basal promoter element important for exclusive RNA polymerase III transcription of the EBER 2 gene.

1993 ◽  
Vol 13 (5) ◽  
pp. 2655-2665 ◽  
Author(s):  
J G Howe ◽  
M D Shu
Keyword(s):  
Rna Polymerase ◽  
Consensus Sequence ◽  
Rna Polymerase Iii ◽  
Class Ii ◽  
Transcription Unit ◽  
Tata Box ◽  
Promoter Element ◽  
Class Iii ◽  
Polymerase Iii

The Epstein-Barr virus-encoded small RNA (EBER) genes are transcribed by RNA polymerase III, but their transcription unit appears to contain both class II and class III promoter elements. One of these promoter element, a TATA-like box which we call the EBER TATA box, or ETAB, is located in a position typical for a class II TATA box but contains G/C residues in the normal T/A motif and a conserved thymidine doublet. Experiments using chloramphenicol acetyltransferase constructs and mutations in the TATA box of the adenovirus major late promoter showed that the ETAB promoter element does not substitute for a class II TATA box. However, when the ETAB promoter element sequence was changed to a class II TATA box consensus sequence, the EBER 2 gene was transcribed in vitro by both RNA polymerases II and III. From these results, we conclude that the ETAB promoter element is important for the exclusive transcription of the EBER 2 gene by RNA polymerase III.

Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III

1994 ◽  
Vol 14 (3) ◽  
pp. 2147-2158
Author(s):  
R J Maraia ◽  
D J Kenan ◽  
J D Keene
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
Class Iii ◽  
Ample Evidence ◽  
Polymerase Iii ◽  
Transcription Complexes ◽  
Pol Iii

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7

1994 ◽  
Vol 14 (9) ◽  
pp. 6164-6170
Author(s):  
P P Sadhale ◽  
N A Woychik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rna Polymerase Iii ◽  
Sodium Dodecyl ◽  
Polymerase Iii ◽  
Conditional Mutant ◽  
5.8S Rrna ◽  
Cell Growth And Viability

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

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