Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome

ABSTRACTTemperature sensitive (TS) mutants have been foundational in the characterization of essential genes. However, a high-throughput workflow for characterization of biophysical changes in TS mutants is lacking. Temperature sensitive Mutant Proteome Profiling (TeMPP) is a novel application of mass spectrometry (MS) based thermal proteome profiling (TPP) to characterize effects of missense mutations on protein stability and PPIs. This study characterizes missense mutations in two different subunits of the 26S proteasome on the thermal stability of the proteome at large, revealing distinct mechanistic details that were not obtained using only steady-state transcriptome and proteome analyses. TeMPP is a precise approach to measure changes in missense mutant containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Overall, TeMPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, non-biased fashion.

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Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.721 ◽

1991 ◽

Vol 11 (2) ◽

pp. 721-730 ◽

Cited By ~ 50

Author(s):

J Y Lee ◽

C E Rohlman ◽

L A Molony ◽

D R Engelke

Keyword(s):

Saccharomyces Cerevisiae ◽

Essential Gene ◽

Rnase P ◽

Single Copy ◽

Temperature Sensitive ◽

Coding Region ◽

Gene Encoding ◽

Processing Steps ◽

Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

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Characterization of the dnaG Locus inMycobacterium smegmatis Reveals Linkage of DNA Replication and Cell Division

Journal of Bacteriology ◽

10.1128/jb.180.1.65-72.1998 ◽

1998 ◽

Vol 180 (1) ◽

pp. 65-72 ◽

Cited By ~ 20

Author(s):

Amy G. Klann ◽

Aimee E. Belanger ◽

Angelica Abanes-De Mello ◽

Janice Y. Lee ◽

Graham F. Hatfull

Keyword(s):

Cell Division ◽

Dna Replication ◽

Mycobacterium Smegmatis ◽

Genomic Library ◽

Essential Gene ◽

Coupled Processes ◽

Temperature Sensitive ◽

Macromolecular Synthesis ◽

Gene Encoding

ABSTRACT We have isolated a UV-induced temperature-sensitive mutant ofMycobacterium smegmatis that fails to grow at 42°C and exhibits a filamentous phenotype following incubation at the nonpermissive temperature, reminiscent of a defect in cell division. Complementation of this mutant with an M. smegmatis genomic library and subsequent subcloning reveal that the defect lies within the M. smegmatis dnaG gene encoding DNA primase. Sequence analysis of the mutant dnaG allele reveals a substitution of proline for alanine at position 496. Thus, dnaG is an essential gene in M. smegmatis, and DNA replication and cell division are coupled processes in this species. Characterization of the sequences flanking the M. smegmatis dnaG gene shows that it is not part of the highly conserved macromolecular synthesis operon present in other eubacterial species but is part of an operon with a dgt gene encoding dGTPase. The organization of this operon is conserved in Mycobacterium tuberculosis andMycobacterium leprae, suggesting that regulation of DNA replication, transcription, and translation may be coordinated differently in the mycobacteria than in other bacteria.

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Molecular genetic characterization of Drosophila ATM conserved functional domains

Genome ◽

10.1139/g10-067 ◽

2010 ◽

Vol 53 (10) ◽

pp. 778-786 ◽

Cited By ~ 11

Author(s):

M. Pedersen ◽

S. Tiong ◽

S. D. Campbell

Keyword(s):

Molecular Mechanisms ◽

Structural Integrity ◽

Molecular Genetic ◽

Telomere Maintenance ◽

Temperature Sensitive ◽

Missense Mutations ◽

Dna Double Strand Breaks ◽

Gene Encoding ◽

Atm Kinase

ATM-related kinases promote repair of DNA double-strand breaks and maintenance of chromosome telomeres, functions that are essential for chromosome structural integrity in all eukaryotic organisms. In humans, loss of ATM function is associated with ataxia telangiectasia, a neurodegenerative disease characterized by extreme sensitivity to DNA damage. Drosophila melanogaster has recently emerged as a useful animal model for analyzing the molecular functions of specific domains of this large, multifunctional kinase. The gene encoding Drosophila ATM kinase (dATM) was originally designated tefu because of the telomere fusion defects observed in atm mutants. In this report, molecular characterization of eight atm (tefu) alleles identified nonsense mutations predicted to truncate conserved C-terminal domains of the dATM protein, as well as two interesting missense mutations. One of these missense mutations localized within a putative HEAT repeat in the poorly characterized N-terminal domain of dATM (atm4), whereas another associated with a temperature-sensitive allele (atm8) changed the last amino acid of the conserved FATC domain. Leveraging this molecular information with the powerful genetic tools available in Drosophila should facilitate future analysis of conserved ATM-mediated molecular mechanisms that are important for telomere maintenance, DNA repair, and neurodegeneration.

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Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.721-730.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 721-730 ◽

Cited By ~ 1

Author(s):

J Y Lee ◽

C E Rohlman ◽

L A Molony ◽

D R Engelke

Keyword(s):

Saccharomyces Cerevisiae ◽

Essential Gene ◽

Rnase P ◽

Single Copy ◽

Temperature Sensitive ◽

Coding Region ◽

Gene Encoding ◽

Processing Steps ◽

Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

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SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.672 ◽

1987 ◽

Vol 7 (2) ◽

pp. 672-678 ◽

Cited By ~ 29

Author(s):

L Neigeborn ◽

J L Celenza ◽

M Carlson

Keyword(s):

Gene Dosage ◽

Essential Gene ◽

Regulatory Region ◽

Upstream Region ◽

Temperature Sensitive ◽

Missense Mutations ◽

Haploid Cell ◽

Recessive Mutations ◽

In Trans ◽

Extragenic Suppressors

Dominant and recessive mutations at the SSN20 locus were previously isolated as extragenic suppressors of mutations in three genes (SNF2, SNF5, and SNF6) that are required in trans to derepress invertase expression. All ssn20 alleles cause recessive, temperature-sensitive lethality. In this study we cloned the SSN20 gene, identified a 4.6-kilobase poly(A)-containing RNA, and showed that disruption of the gene is lethal in a haploid cell. Genetic mapping of SSN20 to a locus on chromosome VII 10 centimorgans distal to cly8 led to the finding that SSN20 is the same gene as SPT6, which affects expression of delta insertions in the 5' noncoding region of HIS4 (F. Winston, D. T. Chaleff, B. Valent, and G. R. Fink, Genetics 107:179-197, 1984). We also showed that an ssn20 mutation restored expression of secreted invertase from deletions of the SUC2 upstream regulatory region; ssn20 restored derepression of SUC2 mRNA in strains with a SUC2 upstream region deletion or a snf2 mutation. Increased or decreased gene dosage of SSN20 also suppressed defects that are suppressed by ssn20 missense mutations. These findings suggest that SSN20 plays a role in general transcriptional processes.

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Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.

Molecular Biology of the Cell ◽

10.1091/mbc.8.1.171 ◽

1997 ◽

Vol 8 (1) ◽

pp. 171-187 ◽

Cited By ~ 64

Author(s):

K Kominami ◽

N Okura ◽

M Kawamura ◽

G N DeMartino ◽

C A Slaughter ◽

...

Keyword(s):

Amino Acids ◽

Essential Gene ◽

Gradient Centrifugation ◽

26S Proteasome ◽

Temperature Sensitive ◽

Functional Studies ◽

Regulatory Module ◽

Acid Protein ◽

Multicopy Suppressors ◽

Genes Encoding

Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome.

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Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid

Applied and Environmental Microbiology ◽

10.1128/aem.70.12.7511-7519.2004 ◽

2004 ◽

Vol 70 (12) ◽

pp. 7511-7519 ◽

Cited By ~ 122

Author(s):

Tamara M. Maier ◽

Andrea Havig ◽

Monika Casey ◽

Francis E. Nano ◽

Dara W. Frank ◽

...

Keyword(s):

High Efficiency ◽

Genetic Manipulation ◽

Temperature Sensitive ◽

Infectious Dose ◽

Francisella Novicida ◽

Shuttle Plasmid ◽

Multiple Routes ◽

Multiple Cloning Sites

ABSTRACT Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 × 107 CFU μg of DNA−1 in F. tularensis LVS, Francisella novicida U112, and E. coli DH5α. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.

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Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease

International Journal of Molecular Sciences ◽

10.3390/ijms21239088 ◽

2020 ◽

Vol 21 (23) ◽

pp. 9088

Author(s):

Daria Biernacka ◽

Patrycja Gorzelak ◽

Gracjana Klein ◽

Satish Raina

Keyword(s):

Elevated Temperatures ◽

Essential Gene ◽

Alternative Pathway ◽

Temperature Sensitive ◽

Isolation And Characterization ◽

Suppressor Mutations ◽

Lipopolysaccharide Biosynthesis ◽

Multicopy Suppressors ◽

Essential Function

We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of ΔlapB revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of ΔlapB by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated lapC, suggesting LapB and LapC act antagonistically. The same lapC gene was identified during selection for mutations that induce transcription from LPS defects-responsive rpoEP3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of lapC mutants that restored growth at elevated temperatures mapped to lapA/lapB, lpxC and ftsH genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in lapC mutants. Interestingly, a lapC deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts.

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SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.672-678.1987 ◽

1987 ◽

Vol 7 (2) ◽

pp. 672-678

Author(s):

L Neigeborn ◽

J L Celenza ◽

M Carlson

Keyword(s):

Gene Dosage ◽

Essential Gene ◽

Regulatory Region ◽

Upstream Region ◽

Temperature Sensitive ◽

Missense Mutations ◽

Haploid Cell ◽

Recessive Mutations ◽

In Trans ◽

Extragenic Suppressors

Dominant and recessive mutations at the SSN20 locus were previously isolated as extragenic suppressors of mutations in three genes (SNF2, SNF5, and SNF6) that are required in trans to derepress invertase expression. All ssn20 alleles cause recessive, temperature-sensitive lethality. In this study we cloned the SSN20 gene, identified a 4.6-kilobase poly(A)-containing RNA, and showed that disruption of the gene is lethal in a haploid cell. Genetic mapping of SSN20 to a locus on chromosome VII 10 centimorgans distal to cly8 led to the finding that SSN20 is the same gene as SPT6, which affects expression of delta insertions in the 5' noncoding region of HIS4 (F. Winston, D. T. Chaleff, B. Valent, and G. R. Fink, Genetics 107:179-197, 1984). We also showed that an ssn20 mutation restored expression of secreted invertase from deletions of the SUC2 upstream regulatory region; ssn20 restored derepression of SUC2 mRNA in strains with a SUC2 upstream region deletion or a snf2 mutation. Increased or decreased gene dosage of SSN20 also suppressed defects that are suppressed by ssn20 missense mutations. These findings suggest that SSN20 plays a role in general transcriptional processes.

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