Brian Rymond
University of Kentucky, Biology, Faculty Member
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Relatively few genes in the yeast Saccharomyces cerevisiae are known to contain intervening sequences. As a group, yeast ribosomal protein genes exhibit a higher prevalence of introns when compared to non-ribosomal protein genes. In an... more
Relatively few genes in the yeast Saccharomyces cerevisiae are known to contain intervening sequences. As a group, yeast ribosomal protein genes exhibit a higher prevalence of introns when compared to non-ribosomal protein genes. In an effort to quantify this bias we have estimated the prevalence of intron sequences among non-ribosomal protein genes by assessing the number of prp2-sensitive mRNAs in an in vitro translation assay. These results, combined with an updated survey of the GenBank DNA database, support an estimate of 2.5% for intron-containing non-ribosomal protein genes. Furthermore, our observations reveal an intriguing distinction between the distributions of ribosomal protein and non-ribosomal protein intron lengths, suggestive of distinct, gene class-specific evolutionary pressures.
Research Interests: Genetics, Biology, Medicine, Saccharomyces cerevisiae, Ribosomal proteins, and 15 moreGene, ribosomal RNA, Introns, Prevalence, GenBank, Fungal genome size, Intron, Two-Dimensional Gel Electrophoresis, Base Sequence, Ribosomal DNA, Poly A, Two dimensional Gel Electrophoresis, Ribosomal Protein, Thallophyta, and Molecular Sequence Data
I. INTRODUCTION The study of pre-mRNA splicing in yeast has enjoyed remarkable success during the past several years (for other recent reviews, see Guthrie 1991; Rosbash and Seraphin 1991; Ruby and Abelson 1991). This is now a mature... more
I. INTRODUCTION The study of pre-mRNA splicing in yeast has enjoyed remarkable success during the past several years (for other recent reviews, see Guthrie 1991; Rosbash and Seraphin 1991; Ruby and Abelson 1991). This is now a mature discipline in which detailed and mechanistic questions are being asked and answered. However, the complexity of the process, i.e., the large number of gene products needed for splicing and the multiple levels at which the problem is currently being studied, can limit the attention of the casual observer. Yet conclusions from these studies in yeast have played an important, if not central, role in our current view of how eukaryotic pre-mRNA processing takes place. As with many other topics covered in these volumes, two facts help to explain the importance of the yeast work on splicing. First, Saccharomyces cerevisiae allows the application of the three major tools of modern molecular biology: genetics, biochemistry, and physiology. This chapter will show that the ability to combine all three approaches has had a major influence on our current understanding of pre-mRNA splicing. Second, yeast splicing and metazoan splicing are very similar and rely on many of the same factors and principles. The apparent differences may reflect the shortcomings of the strictly biochemical approach available to the metazoan workers, rather than genuine differences in the processes. The genetic study of pre-mRNA splicing in S. cerevisiae has as its roots a study that preceded by almost 10 years the discovery of splicing itself. Between 1967 and 1970,...
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ABSTRACT The Saccharomyces cerevisiae branchpoint binding protein (BBP) is a 53 kDa pre-mRNA processing factor with characteristic STAR/GSG protein organization. This includes a central RNA binding site composed of an extended Type I KH... more
ABSTRACT The Saccharomyces cerevisiae branchpoint binding protein (BBP) is a 53 kDa pre-mRNA processing factor with characteristic STAR/GSG protein organization. This includes a central RNA binding site composed of an extended Type I KH domain with an adjacent QUA2 motif. Downstream of KH-QUA2 are two CCHC-type zinc knuckles and a proline-rich C-terminal interaction domain (Fig. 1A). The QUA1 homodimerization motif found upstream of the KH-QUA2 sequence in other STAR/GSG family members is absent in BBP and replaced by a site for the phylogenetically conserved binding partner, Mud2/U2AF65. BBP’s name reflects the fact that it binds the conserved RNA sequence, UACUAAC, called the branchpoint motif found near the 3′ end of yeast introns. This sequence contains the catalytic adenosine (underlined) which directs the first RNA transesterification reaction in splicing chemistry. BBP recruitment to the branchpoint initiates a series of spliceosomal subunit addition and rearrangement events that ultimately configures the active site of this enzyme.1 The mammalian homolog, ZF M1/ZNF162/D11S636/SF1 (henceforth, SF1), was first identified in a screen for genes associated with Type 1 multiple endocrine neoplasia2 and was subsequently shown to act similarly to BBP in mammalian splicing.3,4 BBP/SF1 is essential for viability in organisms spanning the evolutionary spectrum from yeast to Caenorhabditis elegans to mice. In addition, mice heterozygous for a SF1 knockout allele show enhanced susceptibility to azoxymethane-induced colon tumorigenesis5 adding BBP/SF1 to the growing list of RNA processing factors implicated in genetic disease.6 Summarized below is our current understanding of BBP structure and its proposed multifaceted contribution to mRNA biogenesis and function. Reference to SF1 will be made to fill gaps in our understanding of BBP and to highlight areas of clear similarity or difference between yeast and mammals. Figure 1Domain organization of the BBP/SF1 splicing factor. A) Schematic representation of BBP. Indicated are the ULM domain-containing region that mediates Mud2 association (Heterodimerization); the RNA binding element (KH_QUA2), two CCHC zinc knuckles (Zn) and the positions conforming to the Smy2 binding site, PPG{F/I/L/M/V}. The Smy2 sequences overlap a clathrin adaptor appendage domain that has not been scored for function in BBP. B) Alignment of the yeast BBP and human SF1 proteins using Blossom 62 matrix with identities shaded black and conserved residues shaded gray. Shown above BBP is the ULM sub-sequence critical to the SF1/U2AF65 interaction, the location of the KIS phosphorylation motif and the βααββ αα secondary structures (thick dashed line) of the KH-QUA2 motif. Also shown are the two loop sequences that contribute to the RNA binding channel (^) and the six Smy2 binding sites (*).
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A set of gene fusions have been constructed between the transcriptional and translational initiation signals of the yeast CYC1 gene, encoding iso-1-cytochrome c, and the coding sequence of the Escherichia coli galK gene encoding... more
A set of gene fusions have been constructed between the transcriptional and translational initiation signals of the yeast CYC1 gene, encoding iso-1-cytochrome c, and the coding sequence of the Escherichia coli galK gene encoding galactokinase. These fusions are contained on plasmids which have both yeast and E. coli replication origins and selectable markers and, therefore, can be used to transform either yeast or E. coli cells. When galactokinase-deficient (gall-) yeasts were transformed with these plasmids the resulting Gal+ transformants were heterogeneous with respect to their galactokinase levels. The galactokinase levels in all were subject to glucose repression, characteristic of the transcriptional regulation of the CYC1 gene. The fusion points for representative plasmids were determined by DNA sequence analysis, and from these data, the differential expression of the galK gene could be explained. One fusion plasmid, YRpR1, which gave the highest level of galK expression, was characterized further. As an additional demonstration that galactokinase expression from the fusion was under CYC1 transcriptional control, a cis-dominant, CYC1-linked mutation known to drastically reduce CYC1 gene transcription was introduced into YRpR1 and shown to similarly effect galK expression. The galK mRNA produced from the fused gene of YCpR1, a centromere-containing derivative of YRpR1, consisted of the mRNA leader sequence plus the first four codons of the CYC1 gene, the galK coding sequence, then the remainder of the CYC1 coding sequence and the 175 nucleotide non-translated 3' sequence. As a demonstration of the usefulness of these plasmids for the selection of regulatory mutants, two mutants capable of greatly enhanced levels of galactokinase expression were isolated. Preliminary characterization of these mutations indicates that they likewise affect the expression of the chromosomal CYC1 gene.
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Research Interests: Biology, Cell Biology, Medicine, Biological Sciences, Saccharomyces cerevisiae, and 11 moreProtein Structure and Function, Molecular and cellular biology, Sequence alignment, Biogenesis, Protein Complex Detection, Amino Acid Sequence, Base Sequence, Cytoplasm, Molecular Sequence Data, Spliceosome, and Medical and Health Sciences
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Research Interests: Genetics, M Rna Processing, Biology, Cell Cycle, Drosophila melanogaster, and 15 moreMedicine, Biological Sciences, Humans, Sequence alignment, Animals, Physical sciences, Cell Fractionation, Alternative splicing, Proteins, HeLa cells, Relational data, Alternative Splicing, Amino Acid Sequence, Molecular Sequence Data, and Spliceosome
Research Interests: Biochemistry, Bioinformatics, Developmental Biology, Climate Change, Computational Biology, and 15 moreBiotechnology, Cancer, Biology, Cell Cycle, Ecology, Drug Discovery, Astrophysics, Astronomy, DNA, Drug Design, Cell Signalling, Drug Delivery Systems, Antineoplastic Agents, Earth Science, and Biochemistry and cell biology
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The binding of a Ul small nuclear ribonucleoprotein (snRNP) particle to the 5' splice site region of a pre-mRNA is a primary step of intron recognition. In this report, we identify a novel 75-kDa polypeptide of Saccharomyces... more
The binding of a Ul small nuclear ribonucleoprotein (snRNP) particle to the 5' splice site region of a pre-mRNA is a primary step of intron recognition. In this report, we identify a novel 75-kDa polypeptide of Saccharomyces cerevisiae, Prp39p, necessary for the stable interaction of mRNA precursors with the snRNP components of the pre-mRNA splicing machinery. In vivo, temperature inactivation or metabolic depletion of Prp39p blocks pre-mRNA splicing and causes growth arrest. Analyses of cell extracts reveal a specific and dramatic increase in the electrophoretic mobility of the Ul snRNP particle upon Prp39p depletion and demonstrate that extracts deficient in Prp39p activity are unable to form either the CC1 or CC2 commitment complex band characteristic of productive Ul snRNP/pre-mRNA association. Immunological studies establish that Prp39p is uniquely associated with the Ul snRNP and is recruited with the Ul snRNP into splicing complexes. On the basis of these and related obse...
The U2 snRNP promotes prespliceosome assembly through interactions that minimally involve the branchpoint binding protein, Mud2p, and the pre-mRNA. We previously showed that seven proteins copurify with the yeast (Saccharomyces... more
The U2 snRNP promotes prespliceosome assembly through interactions that minimally involve the branchpoint binding protein, Mud2p, and the pre-mRNA. We previously showed that seven proteins copurify with the yeast (Saccharomyces cerevisiae) SF3b U2 subcomplex that associates with the pre-mRNA branchpoint region: Rse1p, Hsh155p, Hsh49p, Cus1p, and Rds3p and unidentified subunits p10 and p17. Here proteomic and genetic studies identify Rcp10p as p10 and show that it contributes to SF3b stability and is necessary for normal cellular Cus1p accumulation and for U2 snRNP recruitment in splicing. Remarkably, only the final 53 amino acids of Rcp10p are essential. p17 is shown to be composed of two accessory splicing factors, Bud31p and Ist3p, the latter of which independently associates with the RES complex implicated in the nuclear pre-mRNA retention. A directed two-hybrid screen reveals a network of prospective interactions that includes previously unreported intra-SF3b contacts and SF3b i...
Research Interests: Genetics, Biology, Proteomics, Medicine, Biological Sciences, and 12 moreSaccharomyces cerevisiae, Molecular and cellular biology, Enzyme, RNA-binding proteins, Amino Acid Profile, Amino Acid Sequence, Protein Binding, Conformational Change, Catalytic Activity, Branch point, Molecular Sequence Data, and Medical and Health Sciences
Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway. Here we show that Rds3p resides in the yeast spliceosome... more
Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway. Here we show that Rds3p resides in the yeast spliceosome and is essential for splicing in vitro. Rds3p purified from yeast stably associates with at least five U2 snRNP proteins, Cus1p, Hsh49p, Hsh155p, Rse1p, and Ist3p/Snu17p, and with the Yra1p RNA export factor. A mutation upstream of the first Rds3p zinc finger causes the conditional release of the putative branchpoint nucleotide binding protein, Ist3p/Snu17p, and weakens Rse1p interaction with the Rds3p complex. The resultant U2 snRNP particle migrates exceptionally slowly in polyacrylamide gels, suggestive of a disorganized structure. U2 snRNPs depleted of Rds3p fail to form stable prespliceosomes, although U2 snRNA stability is not affected. Metabolic depletion of Yra1p blocks cell growth but not splicing, suggesting that Yra1p association with Rd...
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Functional analysis of the Mtl1 protein inSaccharomyces cerevisiaehas revealed that this transmembrane sensor endows yeast cells with resistance to oxidative stress through a signaling mechanism called the cell wall integrity pathway... more
Functional analysis of the Mtl1 protein inSaccharomyces cerevisiaehas revealed that this transmembrane sensor endows yeast cells with resistance to oxidative stress through a signaling mechanism called the cell wall integrity pathway (CWI). We observed upregulation of multiple heat shock proteins (HSPs), proteins associated with the formation of stress granules, and the phosphatase subunit of trehalose 6-phosphate synthase which suggests thatmtl1Δstrains undergo intrinsic activation of a non-lethal heat stress response. Furthermore, quantitative global proteomic analysis conducted on TMT-labeled proteins combined with metabolome analysis revealed thatmtl1Δstrains exhibit decreased levels of metabolites of carboxylic acid metabolism, decreased expression of anabolic enzymes and increased expression of catabolic enzymes involved in the metabolism of amino acids, with enhanced expression of mitochondrial respirasome proteins. These observations support the idea that Mtl1 protein contro...
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Antifungal drug discovery and design is very challenging because of the considerable similarities in genetic features and metabolic pathways between fungi and humans. However, cell wall composition represents a notable point of... more
Antifungal drug discovery and design is very challenging because of the considerable similarities in genetic features and metabolic pathways between fungi and humans. However, cell wall composition represents a notable point of divergence. Therefore, a research strategy was designed to improve our understanding of the mechanisms for maintaining fungal cell wall integrity, and to identify potential targets for new drugs that modulate the underlying protein-protein interactions in Saccharomyces cerevisiae. This study defines roles for Wsc2p and Wsc3p and their interacting protein partners in the cell wall integrity signaling and cell survival mechanisms that respond to treatments with fluconazole and hydrogen peroxide. By combined genetic and biochemical approaches, we report the discovery of 12 novel protein interactors of Wsc2p and Wsc3p. Of these, Wsc2p interacting partners Gtt1p and Yck2p, have opposing roles in the resistance and sensitivity to fluconazole treatments respectively...
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We have cloned and sequenced the yeast SNR19 gene and show here that snR19 is the yeast homolog of metazoan U1 snRNA. sn R19 is 569 nucleotides long, strikingly larger than its metazoan counterpart. The two molecules resemble each other... more
We have cloned and sequenced the yeast SNR19 gene and show here that snR19 is the yeast homolog of metazoan U1 snRNA. sn R19 is 569 nucleotides long, strikingly larger than its metazoan counterpart. The two molecules resemble each other closely in the predicted secondary structure of their first 50 nucleotides. Primary sequence homology is restricted to some of their single-stranded regions, including 11 consecutive nucleotides at the 5' end of the two molecules, the region that interacts with pre-mRNA 5' splice junctions. snR19 is spliceosome-associated and required for in vitro pre-mRNA splicing. We also note that 8 sequences in snR19 have extensive complementarity to snR20, the large yeast U2 RNA, suggesting that yeast U1 may interact with yeast U2 by base-pairing.
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Non-muscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae. Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an... more
Non-muscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae. Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis using the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus a...
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Thesis (Ph. D.)--State University of New York at Albany, Dept. of Biological Sciences, 1984. Includes bibliographical references (leaves 57-64). Microfilm. s
RNase H and synthetic DNA oligonucleotides were used to analyze the ribonucleoprotein (RNP) structure of the yeast spliceosome and to assay the pre-mRNA sequence requirements for step 1 of splicing. The data suggest that tight, stable... more
RNase H and synthetic DNA oligonucleotides were used to analyze the ribonucleoprotein (RNP) structure of the yeast spliceosome and to assay the pre-mRNA sequence requirements for step 1 of splicing. The data suggest that tight, stable contacts between the pre-mRNA and the spliceosome may be limited to the 5' splice site and branch point regions of the intron. A 30 nucleotide segment 3' of the branch point was found to be necessary for spliceosome maturation and essential for step 1 of splicing. Somewhat surprisingly, the 3' splice site was sensitive to nuclease digestion and completely dispensable for step 1 of splicing.
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We have presented the results of our studies of the expression of the CYC genes from plasmids. All our data indicate that the levels of expression and the regulation of expression are very similar for the plasmid-borne genes and the... more
We have presented the results of our studies of the expression of the CYC genes from plasmids. All our data indicate that the levels of expression and the regulation of expression are very similar for the plasmid-borne genes and the chromosomal genes when care is taken to construct the appropriate plasmids. The usefulness of these plasmids has been demonstrated: mutations affecting regulatory sites adjacent to genes of interest have been constructed [such as the Xho I deletion and inversion in the YCpCYC1(2.4) plasmid] and selected [as in the case of the IS1 insertion into the YCpCYC7(2) plasmid], and these mutations have led us to some tentative conclusions about the location and nature of the regulatory sites of these genes. Furthermore, transformation with plasmids containing modified genes or fusions has permitted isolation of genomic regulatory mutants, as in the selection of lac+ suppressors of the lac- CYC1 1/x inversion carried on the YCpCYC1(2.4) 1/x plasmid. Although we ca...
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U4 snRNA release from the spliceosome occurs through an essential but ill-defined Prp38p-dependent step. Here we report the results of a dosage suppressor screen to identify genes that contribute to PRP38 function. Elevated expression of... more
U4 snRNA release from the spliceosome occurs through an essential but ill-defined Prp38p-dependent step. Here we report the results of a dosage suppressor screen to identify genes that contribute to PRP38 function. Elevated expression of a previously uncharacterized gene, SPP381, efficiently suppresses the growth and splicing defects of a temperature-sensitive (Ts) mutant prp38-1. This suppression is specific in that enhanced SPP381 expression does not alter the abundance of intronless RNA transcripts or suppress the Ts phenotypes of other prp mutants. Since SPP381 does not suppress a prp38::LEU2 null allele, it is clear that Spp381p assists Prp38p in splicing but does not substitute for it. Yeast SPP381 disruptants are severely growth impaired and accumulate unspliced pre-mRNA. Immune precipitation studies show that, like Prp38p, Spp381p is present in the U4/U6.U5 tri-snRNP particle. Two-hybrid analyses support the view that the carboxyl half of Spp381p directly interacts with the ...
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Clf1 is a conserved spliceosome assembly factor composed predominately of TPR repeats. Here we show that the TPR elements are not functionally equivalent, with the amino terminus of Clf1 being especially sensitive to change. Deletion and... more
Clf1 is a conserved spliceosome assembly factor composed predominately of TPR repeats. Here we show that the TPR elements are not functionally equivalent, with the amino terminus of Clf1 being especially sensitive to change. Deletion and add-back experiments reveal that the splicing defect associated with TPR removal results from the loss of TPR-specific sequence information. Twelve mutants were found that show synthetic growth defects when combined with an allele that lacks TPR2 (i.e., clf1Delta2). The identified genes encode the Mud2, Ntc20, Prp16, Prp17, Prp19, Prp22, and Syf2 splicing factors and four proteins without established contribution to splicing (Bud13, Cet1, Cwc2, and Rds3). Each synthetic lethal with clf1Delta2 (slc) mutant is splicing defective in a wild-type CLF1 background. In addition to the splicing factors, SSD1, BTS1, and BET4 were identified as dosage suppressors of clf1Delta2 or selected slc mutants. These results support Clf1 function through multiple stages...
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The binding of a U1 small nuclear ribonucleoprotein (snRNP) particle to the 5' splice site region of a pre-mRNA is a primary step of intron recognition. In this report, we identify a novel 75-kDa polypeptide of Saccharomyces... more
The binding of a U1 small nuclear ribonucleoprotein (snRNP) particle to the 5' splice site region of a pre-mRNA is a primary step of intron recognition. In this report, we identify a novel 75-kDa polypeptide of Saccharomyces cerevisiae, Prp39p, necessary for the stable interaction of mRNA precursors with the snRNP components of the pre-mRNA splicing machinery. In vivo, temperature inactivation or metabolic depletion of Prp39p blocks pre-mRNA splicing and causes growth arrest. Analyses of cell extracts reveal a specific and dramatic increase in the electrophoretic mobility of the U1 snRNP particle upon Prp39p depletion and demonstrate that extracts deficient in Prp39p activity are unable to form either the CC1 or CC2 commitment complex band characteristic of productive U1 snRNP/pre-mRNA association. Immunological studies establish that Prp39p is uniquely associated with the U1 snRNP and is recruited with the U1 snRNP into splicing complexes. On the basis of these and related obse...
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Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are... more
Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct function...
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A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting... more
A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the co...