scholarly journals Genetic interaction mapping informs integrative structure determination of protein complexes

Science ◽  
2020 ◽  
Vol 370 (6522) ◽  
pp. eaaz4910 ◽  
Author(s):  
Hannes Braberg ◽  
Ignacia Echeverria ◽  
Stefan Bohn ◽  
Peter Cimermancic ◽  
Anthony Shiver ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structure Determination ◽  
Genetic Interaction ◽  
Protein Complexes ◽  
Point Mutations ◽  
Genetic Interactions ◽  
Cellular Functions ◽  
Cross Links

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.

Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II

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.

scholarly journals Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (10) ◽  
pp. 5771-5779 ◽  
Author(s):  
J. Cale Lennon ◽  
Megan Wind ◽  
Laura Saunders ◽  
M. Benjamin Hock ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutant Cells ◽  
Rna Levels

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.

scholarly journals A Motif Shared by TFIIF and TFIIB Mediates Their Interaction with the RNA Polymerase II Carboxy-Terminal Domain Phosphatase Fcp1p in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (20) ◽  
pp. 7438-7449 ◽  
Author(s):  
Michael S. Kobor ◽  
Lisa D. Simon ◽  
Jim Omichinski ◽  
Guoqing Zhong ◽  
Jacques Archambault ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Binding Motif ◽  
Brct Domain ◽  
Sequence Motifs ◽  
Carboxy Terminal ◽  
Ctd Phosphatase

ABSTRACT Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo.

scholarly journals Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

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.

scholarly journals Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences.

1988 ◽  
Vol 8 (5) ◽  
pp. 2021-2033 ◽  
Author(s):  
D E Ayer ◽  
W S Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
Tata Box ◽  
Base Pairs

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences

1988 ◽  
Vol 8 (5) ◽  
pp. 2021-2033
Author(s):  
D E Ayer ◽  
W S Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
Tata Box ◽  
Base Pairs

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

scholarly journals Genetic Interactions of Spt4-Spt5 and TFIIS With the RNA Polymerase II CTD and CTD Modifying Enzymes in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 487-497 ◽  
Author(s):  
Derek L Lindstrom ◽  
Grant A Hartzog
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Normal Function ◽  
Genetic Interactions ◽  
Elongation Factors ◽  
Repeat Domain ◽  
Ctd Phosphatase ◽  
Rna Polymerase Ii Ctd

Abstract Genetic and biochemical studies have identified many factors thought to be important for transcription elongation. We investigated relationships between three classes of these factors: (1) transcription elongation factors Spt4-Spt5, TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA polymerase II; and (3) protein kinases that phosphorylate the CTD and a phosphatase that dephosphorylates it. We observe that spt4 and spt5 mutations cause strong synthetic phenotypes in combination with mutations that shorten or alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases; and affect the CTD phosphatase Fcp1. We show that Spt5 co-immunoprecipitates with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD. Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the ability of Spt5 to bind RNA polymerase II. We find a similar set of genetic interactions between the CTD, CTD modifying enzymes, and TFIIS. In contrast, an spt16 mutation did not show these interactions. These results suggest that the CTD plays a key role in modulating elongation in vivo and that at least a subset of elongation factors are dependent upon the CTD for their normal function.

scholarly journals The Rpb4 Subunit of RNA Polymerase II Contributes to Cotranscriptional Recruitment of 3′ Processing Factors

2008 ◽  
Vol 28 (6) ◽  
pp. 1883-1891 ◽  
Author(s):  
Vanessa M. Runner ◽  
Vladimir Podolny ◽  
Stephen Buratowski
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Chromatin Immunoprecipitation ◽  
The Core ◽  
Catalytic Active Site ◽  
Cross Links ◽  
Processing Factors

ABSTRACT The RNA polymerase II enzyme from the yeast Saccharomyces cerevisiae is a complex of 12 subunits, Rpb1 to Rpb12. Crystal structures of the full complex show that the polymerase consists of two separable components, a 10-subunit core including the catalytic active site and a heterodimer of the Rpb4 and Rpb7 subunits. To characterize the role of the Rpb4/7 heterodimer during transcription in vivo, chromatin immunoprecipitation was used to examine an rpb4Δ strain for effects on the behavior of the core polymerase as well as recruitment of other protein factors involved in transcription. Rpb4/7 cross-links throughout transcribed regions. Loss of Rpb4 results in a reduction of RNA polymerase II levels near 3′ ends of multiple mRNA genes as well as a decreased association of 3′-end processing factors. Furthermore, loss of Rpb4 results in altered polyadenylation site usage at the RNA14 gene. Together, these results indicate that Rpb4 contributes to proper cotranscriptional 3′-end processing in vivo.

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