Elongator and SPT4/SPT5 complexes as proxy to study RNA polymerase II transcript elongation control of plant development

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

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Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4142-4152.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4142-4152

Author(s):

J Archambault ◽

F Lacroute ◽

A Ruet ◽

J D Friesen

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Structural Integrity ◽

Genetic Interaction ◽

Elongation Factor ◽

Chemical Mutagenesis ◽

Yeast Genome ◽

Transcript Elongation

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

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Causes and consequences of RNA polymerase II stalling during transcript elongation

Nature Reviews Molecular Cell Biology ◽

10.1038/s41580-020-00308-8 ◽

2020 ◽

Vol 22 (1) ◽

pp. 3-21

Author(s):

Melvin Noe Gonzalez ◽

Daniel Blears ◽

Jesper Q. Svejstrup

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcript Elongation

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DBIRD complex integrates alternative mRNA splicing with RNA polymerase II transcript elongation

Nature ◽

10.1038/nature10925 ◽

2012 ◽

Vol 484 (7394) ◽

pp. 386-389 ◽

Cited By ~ 77

Author(s):

Pierre Close ◽

Philip East ◽

A. Barbara Dirac-Svejstrup ◽

Holger Hartmann ◽

Mark Heron ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mrna Splicing ◽

Alternative Mrna Splicing ◽

Transcript Elongation

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Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1405181111 ◽

2014 ◽

Vol 111 (18) ◽

pp. 6642-6647 ◽

Cited By ~ 40

Author(s):

V. Schweikhard ◽

C. Meng ◽

K. Murakami ◽

C. D. Kaplan ◽

R. D. Kornberg ◽

...

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcript Elongation

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Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4214 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4214-4222 ◽

Cited By ~ 31

Author(s):

Y Chen ◽

J Weeks ◽

M A Mortin ◽

A L Greenleaf

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Sequence ◽

Pcr Amplification ◽

Single Strand Conformation Polymorphism ◽

Chain Elongation ◽

Developmental Defect ◽

Genes Encoding ◽

Transcript Elongation

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

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