scholarly journals Displacement of Xenopus transcription factor IIIA from a 5S rRNA gene by a transcribing RNA polymerase.

1991 ◽  
Vol 11 (8) ◽  
pp. 3978-3986 ◽  
Author(s):  
F E Campbell ◽  
D R Setzer
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Internal Control ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Transcription Factor Iiia ◽  
Polymerase Iii ◽  
5S Rrna Gene

In the absence of other components of the RNA polymerase III transcription machinery, transcription factor IIIA (TFIIIA) can be displaced from both strands of its DNA-binding site (the internal control region) on the somatic-type 5S rRNA gene of Xenopus borealis during transcription elongation by bacteriophage T7 RNA polymerase, regardless of which DNA strand is transcribed. Furthermore, substantial displacement is observed after the template has been transcribed only once. Since the complete 5S rRNA transcription complex has previously been shown to remain stably bound to the gene during repeated rounds of transcription by either RNA polymerase III or bacteriophage SP6 RNA polymerase, these results indicate that a factor(s) in addition to TFIIIA is required to create a complex that will remain stably associated with the template during transcription. Thus, transcription complex stability during passage of RNA polymerase cannot be explained solely on the basis of the DNA-binding properties of TFIIIA.

Displacement of Xenopus transcription factor IIIA from a 5S rRNA gene by a transcribing RNA polymerase

1991 ◽  
Vol 11 (8) ◽  
pp. 3978-3986
Author(s):  
F E Campbell ◽  
D R Setzer
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Internal Control ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Transcription Factor Iiia ◽  
Polymerase Iii ◽  
5S Rrna Gene

In the absence of other components of the RNA polymerase III transcription machinery, transcription factor IIIA (TFIIIA) can be displaced from both strands of its DNA-binding site (the internal control region) on the somatic-type 5S rRNA gene of Xenopus borealis during transcription elongation by bacteriophage T7 RNA polymerase, regardless of which DNA strand is transcribed. Furthermore, substantial displacement is observed after the template has been transcribed only once. Since the complete 5S rRNA transcription complex has previously been shown to remain stably bound to the gene during repeated rounds of transcription by either RNA polymerase III or bacteriophage SP6 RNA polymerase, these results indicate that a factor(s) in addition to TFIIIA is required to create a complex that will remain stably associated with the template during transcription. Thus, transcription complex stability during passage of RNA polymerase cannot be explained solely on the basis of the DNA-binding properties of TFIIIA.

DNA methylation inhibits transcription by RNA polymerase III of a tRNA gene, but not of a 5S rRNA gene

FEBS Letters ◽  
1990 ◽  
Vol 269 (2) ◽  
pp. 358-362 ◽  
Author(s):  
Daniel Besser ◽  
Frank Götz ◽  
Kai Schulze-Forster ◽  
Herbert Wagner ◽  
Hans Kröger ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Polymerase Iii ◽  
5S Rrna Gene

Transcription termination by RNA polymerase III: uncoupling of polymerase release from termination signal recognition

1992 ◽  
Vol 12 (5) ◽  
pp. 2260-2272
Author(s):  
F E Campbell ◽  
D R Setzer
Keyword(s):  
Rna Polymerase ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Signal Recognition ◽  
Polymerase Iii ◽  
5S Rrna Gene ◽  
Nascent Rna

Xenopus RNA polymerase III specifically initiates transcription on poly(dC)-tailed DNA templates in the absence of other class III transcription factors normally required for transcription initiation. In experimental analyses of transcription termination using DNA fragments with a 5S rRNA gene positioned downstream of the tailed end, only 40% of the transcribing polymerase molecules terminate at the normally efficient Xenopus borealis somatic-type 5S rRNA terminators; the remaining 60% read through these signals and give rise to runoff transcripts. We find that the nascent RNA strand is inefficiently displaced from the DNA template during transcription elongation. Interestingly, only polymerases synthesizing a displaced RNA terminate at the 5S rRNA gene terminators; when the nascent RNA is not displaced from the template, read-through transcripts are synthesized. RNAs with 3' ends at the 5S rRNA gene terminators are judged to result from authentic termination events on the basis of multiple criteria, including kinetic properties, the precise 3' ends generated, release of transcripts from the template, and recycling of the polymerase. Even though only 40% of the polymerase molecules ultimately terminate at either of the tandem 5S rRNA gene terminators, virtually all polymerases pause there, demonstrating that termination signal recognition can be experimentally uncoupled from polymerase release. Thus, termination is dependent on RNA strand displacement during transcription elongation, whereas termination signal recognition is not. We interpret our results in terms of a two-step model for transcription termination in which polymerase release is dependent on the fate of the nascent RNA strand during transcription elongation.

Transcription factor requirements for in vitro formation of transcriptionally competent 5S rRNA gene chromatin

1990 ◽  
Vol 10 (5) ◽  
pp. 2390-2401
Author(s):  
S J Felts ◽  
P A Weil ◽  
R Chalkley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Active Chromatin ◽  
Polymerase Iii ◽  
Protein Dna Interactions ◽  
5S Rrna Gene

The Saccharomyces cerevisiae 5S rRNA gene was used as a model system to study the requirements for assembling transcriptionally active chromatin in vitro with purified components. When a plasmid containing yeast 5S rDNA was assembled into chromatin with purified core histones, the gene was inaccessible to the yeast class III gene transcription machinery. Preformation of a 5S rRNA gene-TFIIIA complex was not sufficient for the formation of active chromatin in this in vitro system. Instead, a complete transcription factor complex consisting of TFIIIA, TFIIIB, and TFIIIC needed to be formed before the addition of histones in order for the 5S chromatin to subsequently be transcribed by RNA polymerase III. Various 5S rRNA maxigenes were constructed and used for chromatin assembly studies. In vitro transcription from these assembled 5S maxigenes revealed that RNA polymerase III was readily able to transcribe through one, two, or four nucleosomes. However, we found that RNA polymerase III was not able to efficiently transcribe a chromatin template containing a more extended array of nucleosomes. In vivo expression experiments indicated that all in vitro-constructed maxigenes were transcriptionally competent. Analyses of protein-DNA interactions formed on these maxigenes in vivo by indirect end labeling indicated that there are extensive interactions throughout the length of these maxigenes. The patterns of protein-DNA interactions formed on these genes are consistent with these DNAs being assembled into extensive nucleosomal arrays.

scholarly journals Transcription factor requirements for in vitro formation of transcriptionally competent 5S rRNA gene chromatin.

1990 ◽  
Vol 10 (5) ◽  
pp. 2390-2401 ◽  
Author(s):  
S J Felts ◽  
P A Weil ◽  
R Chalkley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Active Chromatin ◽  
Polymerase Iii ◽  
Protein Dna Interactions ◽  
5S Rrna Gene

The Saccharomyces cerevisiae 5S rRNA gene was used as a model system to study the requirements for assembling transcriptionally active chromatin in vitro with purified components. When a plasmid containing yeast 5S rDNA was assembled into chromatin with purified core histones, the gene was inaccessible to the yeast class III gene transcription machinery. Preformation of a 5S rRNA gene-TFIIIA complex was not sufficient for the formation of active chromatin in this in vitro system. Instead, a complete transcription factor complex consisting of TFIIIA, TFIIIB, and TFIIIC needed to be formed before the addition of histones in order for the 5S chromatin to subsequently be transcribed by RNA polymerase III. Various 5S rRNA maxigenes were constructed and used for chromatin assembly studies. In vitro transcription from these assembled 5S maxigenes revealed that RNA polymerase III was readily able to transcribe through one, two, or four nucleosomes. However, we found that RNA polymerase III was not able to efficiently transcribe a chromatin template containing a more extended array of nucleosomes. In vivo expression experiments indicated that all in vitro-constructed maxigenes were transcriptionally competent. Analyses of protein-DNA interactions formed on these maxigenes in vivo by indirect end labeling indicated that there are extensive interactions throughout the length of these maxigenes. The patterns of protein-DNA interactions formed on these genes are consistent with these DNAs being assembled into extensive nucleosomal arrays.

scholarly journals Transcription termination by RNA polymerase III: uncoupling of polymerase release from termination signal recognition.

1992 ◽  
Vol 12 (5) ◽  
pp. 2260-2272 ◽  
Author(s):  
F E Campbell ◽  
D R Setzer
Keyword(s):  
Rna Polymerase ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Signal Recognition ◽  
Polymerase Iii ◽  
5S Rrna Gene ◽  
Nascent Rna

Xenopus RNA polymerase III specifically initiates transcription on poly(dC)-tailed DNA templates in the absence of other class III transcription factors normally required for transcription initiation. In experimental analyses of transcription termination using DNA fragments with a 5S rRNA gene positioned downstream of the tailed end, only 40% of the transcribing polymerase molecules terminate at the normally efficient Xenopus borealis somatic-type 5S rRNA terminators; the remaining 60% read through these signals and give rise to runoff transcripts. We find that the nascent RNA strand is inefficiently displaced from the DNA template during transcription elongation. Interestingly, only polymerases synthesizing a displaced RNA terminate at the 5S rRNA gene terminators; when the nascent RNA is not displaced from the template, read-through transcripts are synthesized. RNAs with 3' ends at the 5S rRNA gene terminators are judged to result from authentic termination events on the basis of multiple criteria, including kinetic properties, the precise 3' ends generated, release of transcripts from the template, and recycling of the polymerase. Even though only 40% of the polymerase molecules ultimately terminate at either of the tandem 5S rRNA gene terminators, virtually all polymerases pause there, demonstrating that termination signal recognition can be experimentally uncoupled from polymerase release. Thus, termination is dependent on RNA strand displacement during transcription elongation, whereas termination signal recognition is not. We interpret our results in terms of a two-step model for transcription termination in which polymerase release is dependent on the fate of the nascent RNA strand during transcription elongation.

scholarly journals Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: inactivation of specific transcription factors.

1987 ◽  
Vol 7 (11) ◽  
pp. 3880-3887 ◽  
Author(s):  
L G Fradkin ◽  
S K Yoshinaga ◽  
A J Berk ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Iii ◽  
Cell Protein ◽  
Polymerase Iii ◽  
Infected Cells

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

scholarly journals DNA binding domain and subunit interactions of transcription factor IIIC revealed by dissection with poliovirus 3C protease.

1996 ◽  
Vol 16 (8) ◽  
pp. 4163-4171 ◽  
Author(s):  
Y Shen ◽  
M Igo ◽  
P Yalamanchili ◽  
A J Berk ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Binding Domain ◽  
Alpha Subunit ◽  
Dna Binding Domain ◽  
Poliovirus Infection ◽  
3C Protease ◽  
Polymerase Iii

Transcription factor IIIC (TFIIIC) is a general RNA polymerase III transcription factor that binds the B-box internal promotor element of tRNA genes and the complex of TFIIIA with a 5S rRNA gene. TFIIIC then directs the binding of TFIIIB to DNA upstream of the transcription start site. TFIIIB in turn directs RNA polymerase III binding and initiation. Human TFIIIC contains five different subunits. The 243-kDa alpha subunit can be specifically cross-linked to B-box DNA, but its sequence does not reveal a known DNA binding domain. During poliovirus infection, TFIIIC is cleaved and inactivated by the poliovirus-encoded 3C protease (3Cpro). Here we analyzed the cleavage of TFIIIC subunits by 3Cpro in vitro and during poliovirus infection of HeLa cells. Analyses of the DNA binding activities of the resulting subcomplexes indicated that an N-terminal 83-kDa domain of the alpha subunit associates with the beta subunit to generate the TFIIIC DNA binding domain. Cleavage with 3Cpro also generated an approximately 125-kDa C-terminal fragment of the alpha subunit which remained associated with the gamma and epsilon subunits.

scholarly journals Maize TF IIIA--the first transcription factor IIIA from monocotyledons. Purification and properties.

1997 ◽  
Vol 44 (3) ◽  
pp. 579-589 ◽  
Author(s):  
E Wyszko ◽  
M Radłowski ◽  
S Bartkowiak ◽  
M Z Barciszewska
Keyword(s):  
Transcription Factor ◽  
Zea Mays L ◽  
Cell Extract ◽  
5S Rrna ◽  
Rrna Gene ◽  
Maize Pollen ◽  
Transcription Factor Iiia ◽  
Purification And Properties ◽  
5S Rrna Gene ◽  
First Time

Purification and properties of transcription factor IIIA (TF IIIA) from maize pollen (Zea mays L.) are presented for the first time. The purified protein has a molecular mass of about 35 kDa and exhibits binding affinity toward both 5S rRNA and 5S rRNA gene. It also facilitates transcription of the 5S rRNA gene in a HeLa cell extract.

Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: inactivation of specific transcription factors

1987 ◽  
Vol 7 (11) ◽  
pp. 3880-3887
Author(s):  
L G Fradkin ◽  
S K Yoshinaga ◽  
A J Berk ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Iii ◽  
Cell Protein ◽  
Polymerase Iii ◽  
Infected Cells

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

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