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.

scholarly journals Cloning and Characterization of Two Evolutionarily Conserved Subunits (TFIIIC102 and TFIIIC63) of Human TFIIIC and Their Involvement in Functional Interactions with TFIIIB and RNA Polymerase III

1999 ◽  
Vol 19 (7) ◽  
pp. 4944-4952 ◽  
Author(s):  
Yng-Ju Hsieh ◽  
Zhengxin Wang ◽  
Robert Kovelman ◽  
Robert G. Roeder
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Structure And Function ◽  
Class Iii ◽  
Tetratricopeptide Repeats ◽  
Helix Loop Helix ◽  
Polymerase Iii ◽  
And Function

ABSTRACT Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that mediates transcription of class III genes through direct recognition of promoters (for tRNA and virus-associated RNA genes) or promoter-TFIIIA complexes (for the 5S RNA gene) and subsequent recruitment of TFIIIB and RNA polymerase III. We describe the cognate cDNA cloning and characterization of two subunits (hTFIIIC63 and hTFIIIC102) that are present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC and are related in structure and function to two yeast TFIIIC subunits (yTFIIIC95 and yTFIIIC131) previously shown to interact, respectively, with the promoter (A box) and with a subunit of yeast TFIIIB. hTFIIIC63 and hTFIIIC102 show parallel in vitro interactions with the homologous human TFIIIB and RNA polymerase III components, as well as additional interactions that may facilitate both TFIIIB and RNA polymerase III recruitment. These include novel interactions of hTFIIIC63 with hTFIIIC102, with hTFIIIB90, and with hRPC62, in addition to the hTFIIIC102–hTFIIIB90 and hTFIIIB90–hRPC39 interactions that parallel the previously described interactions in yeast. As reported for yTFIIIC131, hTFIIIC102 contains acidic and basic regions, tetratricopeptide repeats (TPRs), and a helix-loop-helix domain, and mutagenesis studies have implicated the TPRs in interactions both with hTFIIIC63 and with hTFIIIB90. These observations further document conservation from yeast to human of the structure and function of the RNA polymerase III transcription machinery, but in addition, they provide new insights into the function of hTFIIIC and suggest direct involvement in recruitment of both TFIIIB and RNA polymerase III.

scholarly journals A Novel Upstream RNA Polymerase III Promoter Element Becomes Essential When the Chromatin Structure of the Yeast U6 RNA Gene Is Altered

2001 ◽  
Vol 21 (19) ◽  
pp. 6429-6439 ◽  
Author(s):  
Michael P. Martin ◽  
Valerie L. Gerlach ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Tata Box ◽  
Initiation Factor ◽  
Base Pairs ◽  
Polymerase Iii ◽  
U6 Rna

ABSTRACT The Saccharomyces cerevisiae U6 RNA gene,SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription ofSNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position −30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele ofSNR6 with decreased spacing between the A and B blocks,snr6-Δ42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Δ42 is much more sensitive to mutations in a (dT-dA)7 tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Δ42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription ofSNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)7 tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.

scholarly journals Stable transcription complex on a class III gene in a minichromosome.

1985 ◽  
Vol 5 (1) ◽  
pp. 40-45 ◽  
Author(s):  
A B Lassar ◽  
D H Hamer ◽  
R G Roeder
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Stable Complex ◽  
Trna Genes ◽  
Class Iii ◽  
5S Rna ◽  
Polymerase Iii

We have constructed recombinant simian virus 40 molecules containing Xenopus 5S RNA and tRNA genes. Recombinant minichromosomes containing these genes were isolated to study the interaction of RNA polymerase III transcription factors with these model chromatin templates. Minichromosomes containing a tRNAMet gene can be isolated in a stable complex with transcription factors (IIIB and IIIC) and are active in vitro templates for purified RNA polymerase III. In contrast, minichromosomes containing a 5S RNA gene are refractory to transcription by purified RNA polymerase III in either the absence or the presence of other factors.

scholarly journals Differential Utilization of TATA Box-binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters

2013 ◽  
Vol 288 (38) ◽  
pp. 27564-27570 ◽  
Author(s):  
Neha Verma ◽  
Ko-Hsuan Hung ◽  
Jin Joo Kang ◽  
Nermeen H. Barakat ◽  
William E. Stumph
Keyword(s):  
Rna Polymerase ◽  
Binding Protein ◽  
Rna Polymerase Iii ◽  
Tata Box ◽  
Related Factor ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Tata Box Binding Protein ◽  
In Cells

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459–469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

scholarly journals Maf1p, a Negative Effector of RNA Polymerase III inSaccharomyces cerevisiae

2001 ◽  
Vol 21 (15) ◽  
pp. 5031-5040 ◽  
Author(s):  
Krzysztof Pluta ◽  
Olivier Lefebvre ◽  
Nancy C. Martin ◽  
Wieslaw J. Smagowicz ◽  
David R. Stanford ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Growth Defect ◽  
Class Iii ◽  
Gene Encoding ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Pol Iii

ABSTRACT Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

Stable transcription complex on a class III gene in a minichromosome

1985 ◽  
Vol 5 (1) ◽  
pp. 40-45
Author(s):  
A B Lassar ◽  
D H Hamer ◽  
R G Roeder
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Stable Complex ◽  
Trna Genes ◽  
Class Iii ◽  
5S Rna ◽  
Polymerase Iii

We have constructed recombinant simian virus 40 molecules containing Xenopus 5S RNA and tRNA genes. Recombinant minichromosomes containing these genes were isolated to study the interaction of RNA polymerase III transcription factors with these model chromatin templates. Minichromosomes containing a tRNAMet gene can be isolated in a stable complex with transcription factors (IIIB and IIIC) and are active in vitro templates for purified RNA polymerase III. In contrast, minichromosomes containing a 5S RNA gene are refractory to transcription by purified RNA polymerase III in either the absence or the presence of other factors.

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.

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