scholarly journals Mutations in elongation factor EF-1 alpha affect the frequency of frameshifting and amino acid misincorporation in Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 120 (4) ◽  
pp. 923-934
Author(s):  
M G Sandbaken ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Elongation Factor ◽  
Common Mechanism ◽  
Amino Acid Substitutions ◽  
Protein Amino Acid ◽  
Nonsense Mutations ◽  
Reading Frame

Abstract A mutational analysis of the eukaryotic elongation factor EF-1 alpha indicates that this protein functions to limit the frequency of errors during genetic code translation. We found that both amino acid misincorporation and reading frame errors are controlled by EF-1 alpha. In order to examine the function of this protein, the TEF2 gene, which encodes EF-1 alpha in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine. Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations. DNA sequence analysis identified eight different sites in the EF-1 alpha protein that elevate the frequency of mistranslation when mutated. These sites are located in two different regions of the protein. Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations. These mutations may effect mistranslation by altering the binding or hydrolysis of GTP. Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations. These mutations may alter the binding of aminoacyl-tRNA by EF-1 alpha. The identification of frameshift and nonsense suppressor mutations in EF-1 alpha indicates a role for this protein in limiting amino acid misincorporation and reading frame errors. We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms.

scholarly journals Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

1996 ◽  
Vol 16 (2) ◽  
pp. 475-480 ◽  
Author(s):  
X Mao ◽  
B Schwer ◽  
S Shuman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Yeast Cells ◽  
Terminal Deletion ◽  
Methyltransferase Activity

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.

scholarly journals A Saccharomyces cerevisiae mitochondrial transcription factor, sc-mtTFB, shares features with sigma factors but is functionally distinct.

1995 ◽  
Vol 15 (4) ◽  
pp. 2101-2108 ◽  
Author(s):  
G S Shadel ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Dna Supercoiling ◽  
Normal Function ◽  
Sigma Factors ◽  
Promoter Recognition

In Saccharomyces cerevisiae mitochondria, sc-mtTFB is a 341-amino-acid transcription factor required for initiation of transcription from mitochondrial DNA promoters. Specific transcription in vitro requires only sc-mtTFB and the bacteriophage-related core sc-mtRNA polymerase. Mutational analysis of sc-mtTFB has defined two regions of the protein that are important for normal function both in vivo and in vitro. These regions overlap portions of the protein that exhibit similarity to conserved region 2 of bacterial sigma factors. One mutation in this region of sc-mtTFB (tyrosine 108 to arginine [Y108R]) has a defective phenotype that matches that observed for mutations in the corresponding residue of Bacillus subtilis sigma A and sigma E proteins. However, mutations in the sigma 2.4-like region, including a 5-amino-acid deletion corresponding to crucial promoter-contacting amino acids of sigma factors, did not eliminate the ability of sc-mtTFB to initiate transcription specifically in vitro. This suggests a mechanism of promoter recognition for sc-mtRNA polymerase different from that used by bacterial RNA polymerases. Two mutations in a basic region of sc-mtTFB resulted in defective proteins that were virtually dependent on supercoiled DNA templates in vitro. These mutations may have disrupted a DNA-unwinding function of sc-mtTFB that is only manifested in vitro and is partially rescued by DNA supercoiling.

scholarly journals Genotypic and Phenotypic Analyses of Hepatitis C Virus from Patients Treated with JTK-853 in a Three-Day Monotherapy

2012 ◽  
Vol 57 (1) ◽  
pp. 436-444 ◽  
Author(s):  
Naoki Ogura ◽  
Yukiyo Toyonaga ◽  
Izuru Ando ◽  
Kunihiro Hirahara ◽  
Tsutomu Shibata ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Viral Resistance ◽  
Hcv Rna ◽  
Amino Acid Substitutions ◽  
Sequencing Analysis ◽  
Resistance Mutations ◽  
In The Wild

ABSTRACTJTK-853, a palm site-binding NS5B nonnucleoside polymerase inhibitor, shows antiviral activityin vitroand in hepatitis C virus (HCV)-infected patients. Here, we report the results of genotypic and phenotypic analyses of resistant variants in 24 HCV genotype 1-infected patients who received JTK-853 (800, 1,200, or 1,600 mg twice daily or 1,200 mg three times daily) in a 3-day monotherapy. Viral resistance in NS5B was investigated using HCV RNA isolated from serum specimens from the patients. At the end of treatment (EOT) with JTK-853, the amino acid substitutions M414T (methionine [M] in position 414 at baseline was replaced with threonine [T] at EOT), C445R (cysteine [C] in position 445 at baseline was replaced with arginine [R] at EOT), Y448C/H (tyrosine [Y] in position 448 at baseline was replaced with cysteine [C] or histidine [H] at EOT), and L466F (leucine [L] in position 466 at baseline was replaced with phenylalanine [F] at EOT), which are known to be typical resistant variants of nonnucleoside polymerase inhibitors, were observed in a clonal sequencing analysis. These substitutions were also selected by a treatment with JTK-853in vitro, and the 50% effective concentration of JTK-853 in the M414T-, C445F-, Y448H-, and L466V-harboring replicons attenuated the susceptibility by 44-, 5-, 6-, and 21-fold, respectively, compared with that in the wild-type replicon (Con1). These findings suggest that amino acid substitutions of M414T, C445R, Y448C/H, and L466F are thought to be viral resistance mutations in HCV-infected patients receiving JTK-853 in a 3-day monotherapy.

scholarly journals The Ras/PKA Signaling Pathway May Control RNA Polymerase II Elongation via the Spt4p/Spt5p Complex in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 165 (3) ◽  
pp. 1059-1070
Author(s):  
Susie C Howard ◽  
Arelis Hester ◽  
Paul K Herman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Signaling Pathway ◽  
Elongation Factor ◽  
Ras Signaling ◽  
Elongation Step ◽  
Rna Polymerase Ii Transcription

Abstract The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA. Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. However, the precise target of these Ras effects has remained unknown. This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process. Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control. First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity. Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa. Finally, Spt5p was efficiently phosphorylated by PKA in vitro. Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth. These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription.

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (9) ◽  
pp. 6350-6360
Author(s):  
F Houman ◽  
C Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Essential Gene ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nonsense Mutations ◽  
Carboxy Terminal

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.

Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein

1989 ◽  
Vol 9 (11) ◽  
pp. 5034-5044
Author(s):  
J L Celenza ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Mutational Analysis ◽  
Gene Dosage ◽  
Regulatory System ◽  
Kinase Domain ◽  
Regulatory Function ◽  
Protein Kinase Activity ◽  
Glucose Limitation

The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1711-1721
Author(s):  
E M McIntosh ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Northern Analysis ◽  
Hindiii Site ◽  
Reading Frame ◽  
Hindiii Restriction Site ◽  
Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (4) ◽  
pp. 2091-2103
Author(s):  
S Türkel ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Physiological Control ◽  
Reading Frame ◽  
Negative Region ◽  
Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

scholarly journals TheEscherichia coliβ-Barrel Assembly Machinery Is Sensitized to Perturbations under High Membrane Fluidity

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Kelly M. Storek ◽  
Rajesh Vij ◽  
Dawei Sun ◽  
Peter A. Smith ◽  
James T. Koerber ◽  
...  
Keyword(s):  
Amino Acid ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Membrane Fluidity ◽  
Amino Acid Substitutions ◽  
Inhibitory Antibody ◽  
Critical Determinant ◽  
Gram Negative ◽  
Growth Environment

ABSTRACTIntegral β-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the β-barrel assembly machine (BAM). This essential complex, composed of a β-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulatein vitro. Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of β-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here, we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing the nonessential BAM lipoproteins, BamB, BamC, or BamE. The disruption of BAM reduces BamA activity, increases outer membrane (OM) fluidity, and activates the σEstress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, an increase in the membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and enable the BAM to tolerate these perturbations. Amino acid substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of β-barrel membrane proteins by BamA.IMPORTANCEBamA is an essential component of the β-barrel assembly machine (BAM) in the outer membranes of Gram-negative bacteria. We have used a recently described inhibitory anti-BamA antibody, MAB1, to identify the molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by amino acid substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity, demonstrating that an increase in membrane fluidity sensitizes the cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which β-barrel folding occurs.

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