Yeast suppressor mutations and transfer RNA processing

Nichols, Mark; Willis, Ian M.; Söll, Dieter

doi:10.1016/0076-6879(90)81137-j

Cited by 13 publications

(6 citation statements)

References 41 publications

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“…The Tfc8TAP strain from the yeast TAP-tagged ORF library was engineered to overexpress Brf1 (approximately 50-fold) by substitution of the natNT2 TEF1 promoter cassette (Janke et al, 2004) for the endogenous BRF1 promoter. Whole cell extracts were prepared and chromatographed by size exclusion chromatography (Sephadex 200), cation exchange (Biorex 70, BRα step elution) and anion exchange (DEAE-Sephadex, step elution) to generate TFIIIB and TFIIIC fractions as described in (Nichols et al, 1990). The TFIIIC fraction was incubated in batch with 1 ml IgG Sepharose resin for 6 hours in 10 mM Tris-HCl pH 8, 150 mM NaCl, 10% glycerol, 1 ug/ml protease inhibitors (leupeptin, pepstatin A, aprotonin and 1mM PMSF).…”

Section: Proteinsmentioning

confidence: 99%

Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160

Moir

Lavados

Lee

et al. 2021

Gene

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Mutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperaturesensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast to Pol III leukodystrophy mutations in RPC160 can be overcome by a combinatorial strategy..

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Section: Proteinsmentioning

confidence: 99%

Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160

Moir

Lavados

Lee

et al. 2021

Gene

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“…Although S. pombe suppressor tRNA genes were used to identify pol III promoter elements as well as to analyze tRNA structure-function, the studies were performed using the S. pombe genes in S. cerevisiae (27, 28). Suppressor tRNAs were also used for genetic screens of fission and budding yeast that identified and characterized genes involved in tRNA biogenesis that include the tRNA isopentenyl transferase, Mod5/ sin1 + / tit1 + , the U34 anticodon modification enzyme, s in3 + / Elp3 , the pol III repressor, Maf1, and the tRNA nuclear export factor, Los1/XpoT ((29-38), also see (39); for historical perspective and early suppressor analysis in S. pombe see (28)).…”

Section: Nonsense Suppressionmentioning

confidence: 99%

A methods review on use of nonsense suppression to study 3′ end formation and other aspects of tRNA biogenesis

Rijal

Maraia

Arimbasseri

2015

Gene

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Suppressor tRNAs bear anticodon mutations that allow them to decode premature stop codons in metabolic marker gene mRNAs, that can be used as in vivo reporters of functional tRNA biogenesis. Here, we review key components of a suppressor tRNA system specific to S. pombe and its adaptations for use to study specific steps in tRNA biogenesis. Eukaryotic tRNA biogenesis begins with transcription initiation by RNA polymerase (pol) III. The nascent pre-tRNAs must undergo folding, 5′ and 3′ processing to remove the leader and trailer, nuclear export, and splicing if applicable, while multiple complex chemical modifications occur throughout the process. We review evidence that precursor-tRNA processing begins with transcription termination at the oligo(T) terminator element, which forms a 3′ oligo(U) tract on the nascent RNA, a sequence-specific binding site for the RNA chaperone, La protein. The processing pathway bifurcates depending on a poorly understood property of pol III termination that determines the 3′ oligo(U) length and therefore the affinity for La. We thus review the pol III termination process and the factors involved including advances using gene-specific random mutagenesis by dNTP analogs that identify key residues important for transcription termination in certain pol III subunits. The review ends with a ‘technical approaches’ section that includes a parts lists of suppressor-tRNA alleles, strains and plasmids, and graphic examples of its diverse uses.

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“…To test the ability of putative S.pombe TFIIIA to support transcription of the 5S rRNA gene, an in vitro transcription system from S.pombe was developed. A whole cell extract was prepared from S.pombe, based upon a method developed for the analysis of the S.pombe RNA polymerase I machinery (33)(34)(35) and used previously to study the transcription of the S.pombe 7S L gene (36). This extract is capable of transcribing a plasmid-borne 5S ribosomal RNA gene in vitro, with or without added recombinant TFIIIA (Fig.…”

Section: Transcriptional Activity Of Putative Spombe Tfiiiamentioning

confidence: 99%

Identification and characterization of transcription factor IIIA from Schizosaccharomyces pombe

Schulman

Setzer

2002

Nucleic Acids Research

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Transcription factor IIIA (TFIIIA) is specifically required for transcription of 5S rRNA genes and is the archetypal C2H2 zinc finger protein. All known vertebrate TFIIIAs have a similar organization: nine zinc fingers, followed by a C-terminal domain of unknown structure. The zinc fingers of Saccharomyces cerevisiae TFIIIA are interrupted between fingers eight and nine by an 81-amino acid spacer. Aside from the amino acids required for zinc finger folding, TFIIIAs from different species are remarkably divergent, whereas the natural binding site, the internal control region of the 5S rRNA gene, is well conserved. We now describe the identification and characterization of TFIIIA from Schizosaccharomyces pombe. This protein is organized differently from its known homologs, in that it contains eight closely spaced zinc fingers, a ninth zinc finger missing a C-terminal Zn2+-coordinating histidine, a 53- amino acid spacer, and an unprecedented tenth zinc finger. We have confirmed the identity of this divergent protein as TFIIIA by showing that it binds specifically and with high affinity to the S.pombe 5S rRNA gene. Comparison of DNase I protection patterns produced by TFIIIA from multiple species suggests a novel mode of DNA recognition by the S.pombe protein. Recombinant S.pombe TFIIIA was also shown to support specific transcription of the 5S rRNA gene in vitro.

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Yeast suppressor mutations and transfer RNA processing

Cited by 13 publications

References 41 publications

Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160

Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160

A methods review on use of nonsense suppression to study 3′ end formation and other aspects of tRNA biogenesis

Identification and characterization of transcription factor IIIA from Schizosaccharomyces pombe

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