scholarly journals The Mof2/Sui1 Protein Is a General Monitor of Translational Accuracy

1998 ◽  
Vol 18 (3) ◽  
pp. 1506-1516 ◽  
Author(s):  
Ying Cui ◽  
Jonathan D. Dinman ◽  
Terri Goss Kinzy ◽  
Stuart W. Peltz
Keyword(s):  
Site Selection ◽  
Start Site ◽  
Wild Type ◽  
Virus Propagation ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Translational Accuracy ◽  
Its Gene ◽  
Killer Virus

ABSTRACT Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed −1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene.mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed −1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.

scholarly journals Ribosomal Protein L3 Mutants Alter Translational Fidelity and Promote Rapid Loss of the Yeast Killer Virus

1999 ◽  
Vol 19 (1) ◽  
pp. 384-391 ◽  
Author(s):  
Stuart W. Peltz ◽  
Amy B. Hammell ◽  
Ying Cui ◽  
Jason Yasenchak ◽  
Lara Puljanowski ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Missense Mutations ◽  
Ribosomal Frameshift ◽  
Virus Propagation ◽  
Particle Assembly ◽  
Ribosomal Frameshifting ◽  
Peptidyl Transfer ◽  
Yeast Killer ◽  
Peptidyltransferase Center ◽  
Killer Virus

ABSTRACT Programmed −1 ribosomal frameshifting is utilized by a number of RNA viruses as a means of ensuring the correct ratio of viral structural to enzymatic proteins available for viral particle assembly. Altering frameshifting efficiencies upsets this ratio, interfering with virus propagation. We have previously demonstrated that compounds that alter the kinetics of the peptidyl-transfer reaction affect programmed −1 ribosomal frameshift efficiencies and interfere with viral propagation in yeast. Here, the use of a genetic approach lends further support to the hypothesis that alterations affecting the ribosome’s peptidyltransferase activity lead to changes in frameshifting efficiency and virus loss. Mutations in theRPL3 gene, which encodes a ribosomal protein located at the peptidyltransferase center, promote approximately three- to fourfold increases in programmed −1 ribosomal frameshift efficiencies and loss of the M1 killer virus of yeast. Themak8-1 allele of RPL3 contains two adjacent missense mutations which are predicted to structurally alter the Mak8-1p. Furthermore, a second allele that encodes the N-terminal 100 amino acids of L3 (called L3Δ) exerts atrans-dominant effect on programmed −1 ribosomal frameshifting and killer virus maintenance. Taken together, these results support the hypothesis that alterations in the peptidyltransferase center affect programmed −1 ribosomal frameshifting.

scholarly journals Altered RNA Editing in Mice Lacking ADAR2 Autoregulation

2006 ◽  
Vol 26 (2) ◽  
pp. 480-488 ◽  
Author(s):  
Yi Feng ◽  
Christopher L. Sansam ◽  
Minati Singh ◽  
Ronald B. Emeson
Keyword(s):  
Alternative Splicing ◽  
Protein Expression ◽  
Translation Termination ◽  
Editing Site ◽  
Wild Type ◽  
Complementary Sequence ◽  
Double Stranded Rna ◽  
Reading Frame ◽  
Site Selective

ABSTRACT ADAR2 is a double-stranded-RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-selective conversion of adenosine to inosine. Previous studies from our laboratory have demonstrated that ADAR2 can modify its own pre-mRNA to create a proximal 3′ splice site containing a noncanonical adenosine-inosine dinucleotide. Alternative splicing to this proximal acceptor adds 47 nucleotides to the mature ADAR2 transcript, thereby resulting in the loss of functional ADAR2 protein expression due to premature translation termination in an alternate reading frame. To examine whether the editing of ADAR2 transcripts represents a negative autoregulatory strategy to modulate ADAR2 protein expression, we have generated genetically modified mice in which the ability of ADAR2 to edit its own pre-mRNA has been selectively ablated by deletion of a critical sequence (editing site complementary sequence [ECS]) required for adenosine-to-inosine conversion. Here we demonstrate that ADAR2 autoediting and subsequent alternative splicing are abolished in homozygous ΔECS mice and that ADAR2 protein expression is increased in numerous tissues compared to wild-type animals. The observed increases in ADAR2 protein expression correlate with the extent of ADAR2 autoediting observed with wild-type tissues and correspond to increases in the editing of ADAR2 substrates, indicating that ADAR2 autoediting is a key regulator of ADAR2 protein expression and activity in vivo.

scholarly journals Core Promoter-Dependent TFIIB Conformation and a Role for TFIIB Conformation in Transcription Start Site Selection

2002 ◽  
Vol 22 (19) ◽  
pp. 6697-6705 ◽  
Author(s):  
Jennifer A. Fairley ◽  
Rachel Evans ◽  
Nicola A. Hawkes ◽  
Stefan G. E. Roberts
Keyword(s):  
Transcription Start Site ◽  
Site Selection ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Initiation Site ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
General Transcription Factor ◽  
Selection Of

ABSTRACT The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5′ and 3′ parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.

scholarly journals Synthetic enhancement of a TFIIB defect by a mutation in SSU72, an essential yeast gene encoding a novel protein that affects transcription start site selection in vivo.

1996 ◽  
Vol 16 (4) ◽  
pp. 1557-1566 ◽  
Author(s):  
Z W Sun ◽  
M Hampsey
Keyword(s):  
Site Selection ◽  
Zinc Binding ◽  
Binding Motif ◽  
Start Site ◽  
Transcription Start ◽  
Gene Encoding ◽  
N Terminus ◽  
Zinc Binding Motif ◽  
Novel Protein

An ssu72 mutant of Saccharomyces cerevisiae was identified as an enhancer of a TFIIB defect (sua7-1) that confers both a cold-sensitive growth defect and a downstream shift in transcription start site selection. The ssu72-1 allele did not affect cold sensitivity but, in combination with sua7-1, created a heat-sensitive phenotype. Moreover, start site selection at the ADH1 gene was dramatically shifted further downstream of the normal sites. Both of these effects could be rescued by either SUA7 or SSU72, thereby defining a functional relationship between the two genes. SSU72 is a single-copy, essential gene encoding a novel protein of 206 amino acids. The ssu72-1 allele is the result of a 30-bp duplication creating a sequence encoding a Cys-X2-Cys-X6-Cys-X2-Cys zinc binding motif near the N terminus of Ssu72p. Mutational analysis demonstrated that the N terminus of Ssu72p is essential for function and that cysteine residues in both the normal and mutant proteins are critical. We discuss the possibility that the potential zinc binding motif of Ssu72 facilitates assembly of the transcription preinitiation complex and that this effect is important for accurate start site selection in vivo.

scholarly journals Ni2+ Transport and Accumulation inRhodospirillum rubrum

1999 ◽  
Vol 181 (15) ◽  
pp. 4554-4560 ◽  
Author(s):  
Richard K. Watt ◽  
Paul W. Ludden
Keyword(s):  
Gene Products ◽  
Wild Type ◽  
Divalent Metal Ions ◽  
Co Dehydrogenase ◽  
Reading Frame ◽  
Carbonyl Cyanide ◽  
Transport Assay ◽  
Fold Excess ◽  
Ni Accumulation

ABSTRACT The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni2+ transport assay was used to monitor uptake and accumulation of 63Ni2+ into R. rubrum and to observe the effect of mutations in thecooC, cooT, and cooJ genes on63Ni2+ transport and accumulation. Cells grown either in the presence or absence of CO transported Ni2+with a Km of 19 ± 4 μM and aV max of 310 ± 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni2+ the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn2+, Mg2+, Ca2+, and Zn2+ showed no inhibition of63Ni2+ transport but Co2+, Cd2+, and Cu2+ inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4°C), by protonophores (carbonyl cyanidem-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N,N′-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni2+ transport. 63Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated 63Ni2+ accumulation coincided with the appearance of CODH activity in the culture, suggesting that the 63Ni2+ was accumulating in CODH. The cooC, cooT, and cooJgenes are required for the increased 63Ni2+accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated63Ni2+ like cells unexposed to CO.

scholarly journals 5 S rRNA is involved in fidelity of translational reading frame.

Genetics ◽  
1995 ◽  
Vol 141 (1) ◽  
pp. 95-105
Author(s):  
J D Dinman ◽  
R B Wickner
Keyword(s):  
Rdna Locus ◽  
Wild Type ◽  
Lacz Reporter ◽  
Lacz Expression ◽  
Lacz Reporter Gene ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Episomal Vector ◽  
Electrophoretic Mobilities ◽  
Insertion Mutations

Abstract Chromosomal mutants (maintenance of frame = mof) in which the efficiency of -1 ribosomal frameshifting is increased can be isolated using constructs in which lacZ expression is dependent upon a -1 shift of reading frame. We isolate a new mof mutation, mof9, in Saccharomyces cerevisiae and show that it is complemented by both single and multi-copy 5 S rDNA clones. Two independent insertion mutations in the rDNA locus (rDNA::LEU2 and rDNA::URA3) also display the Mof- phenotype and are also complemented by single and multi-copy 5 S rDNA clones. Mutant 5 S rRNAs expressed from a plasmid as 20-50% of total 5 S rRNA in a wild-type host also induced the Mof- phenotype. The increase in frameshifting is greatest when the lacZ reporter gene is expressed on a high copy, episomal vector. No differences were found in 5 S rRNA copy number or electrophoretic mobilities in mof9 strains. Both mof9 and rDNA::LEU2 increase the efficiency of +1 frameshifting as well but have no effect on readthrough of UAG or UAA termination codons, indicating that not all translational specificity is affected. These data suggest a role for 5 S rRNA in the maintenance of frame in translation.

scholarly journals The membrane associated accessory protein is an adeno-associated viral egress factor

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zachary C. Elmore ◽  
L. Patrick Havlik ◽  
Daniel K. Oh ◽  
Leif Anderson ◽  
George Daaboul ◽  
...  
Keyword(s):  
Extracellular Vesicle ◽  
Lytic Infection ◽  
Open Reading Frame ◽  
Accessory Protein ◽  
Start Site ◽  
Wild Type ◽  
Reading Frame ◽  
Viral Egress ◽  
Plausible Mechanism ◽  
Multiple Processing

AbstractAdeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with MAAP restored secretion of multiple AAV/MAAPΔ serotypes. Further, multiple processing and analytical methods corroborate that one plausible mechanism by which MAAP promotes viral egress is through AAV/EV association. In addition to characterizing a novel viral egress factor, we highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.

scholarly journals pbpB, a Gene Coding for a Putative Penicillin-Binding Protein, Is Required for Aerobic Nitrogen Fixation in the Cyanobacterium Anabaena sp. Strain PCC7120

2001 ◽  
Vol 183 (2) ◽  
pp. 628-636 ◽  
Author(s):  
Sara Lázaro ◽  
Francisca Fernández-Piñas ◽  
Eduardo Fernández-Valiente ◽  
Amaya Blanco-Rivero ◽  
Francisco Leganés
Keyword(s):  
Sequence Similarity ◽  
Shuttle Vector ◽  
Molecular Size ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Reading Frame ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Peptidoglycan Layer

ABSTRACT Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequencedAnabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and theEscherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal aspbpB and its product as PBP2. The wild-type form ofpbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N2, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.

scholarly journals A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth.

1988 ◽  
Vol 8 (8) ◽  
pp. 3094-3103 ◽  
Author(s):  
C K Shih ◽  
R Wagner ◽  
S Feinstein ◽  
C Kanik-Ennulat ◽  
N Neff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Atp Synthase ◽  
Resistance Mutation ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Reading Frame ◽  
F0f1 Atp Synthase ◽  
General Inhibitor

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

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