RESUMEN
The signal recognition particle (SRP) is a cytoplasmic RNA-protein complex that targets proteins to the rough endoplasmic reticulum. Although SRP functions in the cytoplasm, RNA microinjection and cDNA transfection experiments in animal cells, as well as genetic analyses in yeast, have indicated that SRP assembles in the nucleus. Nonetheless, the mechanisms responsible for nuclear-cytoplasmic transport of SRP RNA and SRP proteins are largely unknown. Here we show that the 19 kDa protein subunit of mammalian SRP, SRP19, was efficiently imported into the nucleus in vitro by two members of the importin beta superfamily of transport receptors, importin 8 and transportin; SRP19 was also imported less efficiently by several other members of the importin beta family. Although transportin is known to import a variety of proteins, SRP19 import is the first function assigned to importin 8. Furthermore, we show that a significant pool of endogenous SRP19 is located in the nucleus, as well as the nucleolus. Our results show that at least one mammalian SRP protein is specifically imported into the nucleus, by members of the importin beta family of transport receptors, and the findings add additional evidence for nuclear assembly of SRP.
Asunto(s)
Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Proteína de Unión al GTP ran/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/química , Células HeLa , Humanos , Carioferinas/análisis , Proteínas Nucleares/análisis , Receptores Citoplasmáticos y Nucleares , Partícula de Reconocimiento de Señal/análisis , beta Carioferinas/análisis , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/análisisRESUMEN
The signal recognition particle (SRP) is a ribonucleoprotein complex that mediates translocation of proteins into the endoplasmic reticulum. Protein SRP19 is an essential structural component of SRP and is believed to promote assembly of the particle. In order to have a convenient source for the purification of milligram amounts of SRP19, we expressed in Escherichia coli a human SRP19 cDNA with an amino-terminal addition of six histidine residues. Expression at 25 degrees C eliminated formation of insoluble SRP19 and resulted in accumulation of soluble hexahistidine-SRP19 to 68% of total cell protein after 24 h. Metal chelation chromatography yielded 40 mg of hexahistidine-SRP19 per liter of culture, with a purity slightly greater than 97%. To examine protein function, the RNA-binding properties of the purified protein were determined by RNA electromobility shift assays. The histidine-tagged SRP19 bound specifically to a 150-nucleotide RNA derived from SRP RNA, with an apparent Kd of 1 nM, and bound, with greatly reduced affinity, to a mutagenized form of the SRP RNA derivative that contained an altered helix 6 tetranucleotide loop. The purified protein was also photochemically crosslinked to the 150-nucleotide SRP RNA fragment, providing the means to potentially identify portions of hexahistidine-SRP19 which are in close proximity to the RNA molecule.
Asunto(s)
Histidina , Proteínas de Unión al ARN/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Reactivos de Enlaces Cruzados , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Oligopéptidos/genética , Péptidos/genética , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/aislamiento & purificación , Rayos UltravioletaRESUMEN
The signal recognition particle is a cytoplasmic RNA-protein complex that mediates translocation of secretory polypeptides into the endoplasmic reticulum. We have used a Xenopus oocyte microinjection assay to determine how signal recognition particle (SRP) RNA is exported from the nucleus. Following nuclear injection, SRP RNA accumulated in the cytoplasm while cytoplasmically injected SRP RNA did not enter the nucleus. Cytoplasmic accumulation of SRP RNA was an apparently facilitated process dependent on limiting trans-acting factors, since nuclear export exhibited saturation kinetics and was completely blocked either at low temperature or by wheat germ agglutinin, a known inhibitor of nuclear pore-mediated transport. At least one target for trans-acting factors that promote nuclear export of SRP RNA appears to be the Alu element of the molecule, since a transcript consisting of only the Alu sequence was exported from the nucleus in a temperature-dependent manner and the Alu transcript competed in the nucleus for transport with intact SRP RNA. Although the identities of trans-acting factors responsible for SRP RNA transport are at present unknown, we suggest that proteins contained within the cytoplasmic form of SRP are candidates. Consistent with this idea were the effects of a mutation in SRP RNA that prevented binding of two known SRP proteins to the Alu sequence.
Asunto(s)
Núcleo Celular/metabolismo , ARN Nuclear Pequeño/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Partícula de Reconocimiento de Señal/metabolismo , Animales , Secuencia de Bases , Transporte Biológico , Citoplasma/metabolismo , Microinyecciones , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Conformación de Ácido Nucleico , Oocitos , Señales de Clasificación de Proteína/metabolismo , ARN/administración & dosificación , ARN/metabolismo , ARN Nuclear Pequeño/química , Proteínas de Unión al ARN/metabolismo , Temperatura , Xenopus laevisRESUMEN
To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei. Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact. Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus. Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible. Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G. W., R. S. Haltiwanger, G. D. Holt, and W. G. Kelly. 1989. Annu. Rev. Biochem. 58:841-874). Although GlcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus. Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures.
Asunto(s)
Núcleo Celular/metabolismo , Oocitos/metabolismo , Ribosomas/metabolismo , Adenosina Trifosfato/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Centrifugación Zonal , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Femenino , Cinética , Sustancias Macromoleculares , Microinyecciones , Membrana Nuclear/fisiología , Saccharomyces cerevisiae , Temperatura , Aglutininas del Germen de Trigo/farmacología , Xenopus laevisRESUMEN
Two particular seven-amino-acid segments from yeast ribosomal protein L29 caused a non-nuclear reporter protein to associate almost exclusively with the yeast nucleus. The two L29-derived nuclear localizing sequences were identical in five of the seven residues, many of which were basic amino acids. Generally, localization of the reporter protein was most impaired by replacement of the basic residues. A particular Arg residue was unique; substitution by any amino acid including Lys diminished nuclear localization of the reporter protein. In L29 the corresponding Arg 25----Lys substitution within the nuclear localizing sequence distal to the N-terminus was without effect, as evidence by normal rates of ribosome assembly and cell growth. However, the analogous Arg 8----Lys substitution within the localizing sequence proximal to the N-terminus led to greatly reduced rates of ribosome assembly and cell growth. Finally, when both localizing sequences contained the Arg----Lys substitution a still greater decrease in ribosome assembly and cell growth was observed. These results were as expected if the two short peptide sequences functioned in nuclear localization and/or assembly of yeast ribosomal protein L29.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/análisis , Secuencia de Aminoácidos , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Datos de Secuencia Molecular , Mutación , Plásmidos , Precursores del ARN/metabolismo , ARN Ribosómico/metabolismo , Proteínas Recombinantes de Fusión , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/ultraestructura , Relación Estructura-Actividad , beta-Galactosidasa/genéticaRESUMEN
A cDNA clone of mouse ribosomal protein L27' was shown previously to be 62% identical in amino acid residues to yeast ribosomal protein L29. The L27' cDNA was expressed in yeast to determine the ability of the mouse protein to substitute for yeast L29 in assembling a functional ribosome. In a yeast strain resistant to cycloheximide by virtue of a recessive mutation in the L29 protein, the murine cDNA did not produce a sensitive phenotype, indicating failure of the mouse L27' protein to assemble into yeast ribosomes. However, when the mouse L27' gene was expressed in cells devoid of L29 and otherwise inviable, the murine protein supported normal growth, demonstrating that mouse ribosomal protein L27' indeed was interchangeable with yeast L29. We conclude that mouse ribosomal protein L27' is assembled into ribosomes in yeast, but yeast L29 is assembled preferentially when both L29 and L27' are present in the same cell.
Asunto(s)
Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Animales , Cicloheximida/farmacología , Farmacorresistencia Microbiana , Genes , Genes Fúngicos , Prueba de Complementación Genética , Ratones , Hibridación de Ácido Nucleico , Plásmidos , Biosíntesis de Proteínas , ARN Mensajero/genética , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
The DNA sequence UAST (TCGTTTTGTACGTTTTTCA) was found to mediate transcription of yeast ribosomal protein gene TCM1. UAST was defined as a transcriptional activator on the basis of loss of transcription accompanying deletions of all or part of UAST, orientation-independent restoration of transcription promoted by a synthetic UAST oligomer inserted either into TCM1 or into the yeast CYC1 gene lacking its transcriptional activation region, and diminished transcription following nucleotide alterations in UAST. UAST bound in vitro to a protein denoted TAF (TCM1 activation factor); TAF was concluded to be a transcriptional activator protein because nucleotide alterations in UAST that diminished transcription in vivo also diminished TAF binding in vitro. The sequence of UAST bore no obvious resemblance to UASrpg, the principal cis-acting element common to most yeast ribosomal protein genes. Likewise, TAF was distinguished from the UASrpg-binding protein TUF, since (i) TAF and TUF were chromatographically separable, (ii) binding of either TAF or TUF to its corresponding UAS was unaffected by an excess of UASrpg or UAST DNA, respectively, and (iii) photochemical cross-linking experiments showed that TAF was a protein of 147 kilodaltons (kDa), while TUF was detected as an approximately 120-kDa polypeptide, consistent with its known size. Cross-linking experiments also revealed that both UAST and UASrpg bound a second heretofore unobserved 82-kDa protein; binding of this additional protein appeared to require binding of TAF or TUF. On the basis of the biochemical characterization of TAF and a lack of sequence similarity between UAST and UASrpg, we suggest that transcription of TCM1 is mediated by a cis-acting sequence and at least one trans-acting factor different from the elements which promote transcription of most other ribosomal protein genes. A second trans-acting factor may be shared by TCM1 and other ribosomal protein genes; this factor could mediate coordinate regulation of these genes.
Asunto(s)
ADN de Hongos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/fisiología , Transcripción Genética , Secuencia de Bases , Reactivos de Enlaces Cruzados , Análisis Mutacional de ADN , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , Regulación de la Expresión Génica , Heparina/metabolismo , Regiones Promotoras GenéticasRESUMEN
When present in excess, the mRNAs for Saccharomyces cerevisiae ribosomal proteins L3 and L29 are translated less efficiently, so that synthesis of these proteins remains commensurate with that of other ribosomal proteins (N.J. Pearson, H.M. Fried, and J.R. Warner, Cell 29:347-355, 1982; J.R. Warner, G. Mitra, W.F. Schwindinger, M. Studeny, and H.M. Fried, Mol. Cell. Biol. 5:1512-1521, 1985). We used a yeast strain with a conditionally transcribed derivative of the L3 gene to deplete cells progressively of L3 mRNA. In this case translation of L3 mRNA did not become more efficient so that L3 was not maintained at a normal level. Even when there was an initial excess of L3 mRNA, interruption of its further transcription produced an immediate drop in L3 synthesis, suggesting that the translational efficiency of preexisting mRNA cannot be altered. Lack of L3 synthesis afforded an opportunity to examine coordinate accumulation of other ribosomal proteins. Without L3, apparent synthesis of several 60S subunit proteins diminished, and 60S subunits did not assemble. A similar phenomenon occurred when, in a second strain, synthesis of ribosomal protein L29 was prevented. Loss of 60S subunit assembly was accompanied by a destabilization of some 60S ribosomal protein mRNAs. These data suggest that synthesis of some S. cerevisiae ribosomal proteins may be regulated posttranscriptionally as a function of the extent to which they are assembled.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Cinética , ARN Mensajero/metabolismo , Proteína Ribosomal L3 , Proteínas Ribosómicas/metabolismo , Transcripción GenéticaRESUMEN
We wished to determine whether there is any specific sequence downstream of the start point of the SP6 promoter which is required for its function in the plasmid pSP64 (Melton et al., 1984). Lack of such specificity would permit in vitro synthesis of an RNA molecule having a 5'-terminal sequence identical to its wild-type in vivo counterpart. To test its requirement, we replaced all of the SP6 sequence downstream of the transcription start point with heterologous nucleotides (nt) and found that any sequence will suffice to permit efficient and accurate transcription. These results permitted construction of plasmids for synthesis of 'authentic' transcripts from cloned DNA. In one case, by an oligodeoxynucleotide-mediated site-specific deletion, we placed the start point of yeast gene TCM1 at nt + 2 of the SP6 promoter and produced in vitro TCM1 mRNA with a wild-type 5'-terminal sequence. We also constructed a vector, pSP64 delta 1, in which the SalI/AccI/HincII recognition sites of pSP64 reside at nt + 2 through + 7. Plasmids such as pSP64 delta 1 may be more useful in some cases as insertion of any DNA fragment at one of these three sites will yield a transcript in which only two to four nt are derived from the vector.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , ADN/metabolismo , Plásmidos , Fagos de Salmonella/genética , Salmonella typhimurium/genética , Transcripción Genética , Secuencia de Bases , Deleción Cromosómica , Clonación Molecular , Enzimas de Restricción del ADN , Conformación de Ácido Nucleico , Operón , Biosíntesis de Proteínas , Fagos de Salmonella/enzimología , Salmonella typhimurium/enzimología , Especificidad de la EspecieRESUMEN
To identify a signal involved in transporting a ribosomal protein to the nucleus, we constructed hybrid genes encoding amino-terminal segments of yeast ribosomal protein L3 joined to the amino-terminal end of the entire Escherichia coli beta-galactosidase molecule. The subcellular locations of the corresponding hybrid proteins in yeast were determined by in situ immunofluorescence. The first 21 amino acids of L3 were sufficient to localize beta-galactosidase to the nucleus. This region shows limited homology to portions of other nuclear proteins identified as essential for their transport. Larger fusion proteins were also localized to the nucleus. However, a hybrid protein containing all but the 14 carboxyl-terminal amino acids from L3 initially failed to localize; this defect was corrected by inserting a glycine- and proline-containing bridge between the L3 and beta-galactosidase moieties. The renovated protein was able to associate with ribosomes, suggesting that, in addition to entering the nucleus, this hybrid polypeptide was assembled into 60S ribosomal subunits that were subsequently exported to the cytoplasm.
Asunto(s)
Proteínas Fúngicas/metabolismo , Señales de Clasificación de Proteína/genética , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico Activo , Núcleo Celular/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/genética , Conformación Proteica , Proteínas Recombinantes/análisis , Proteína Ribosomal L3 , Proteínas Ribosómicas/genética , Homología de Secuencia de Ácido Nucleico , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The rate of accumulation of each ribosomal protein is carefully regulated by the yeast cell to provide the equimolar ratio necessary for the assembly of the ribosome. The mechanisms responsible for this regulation have been examined by introducing into the yeast cell extra copies of seven individual ribosomal protein genes carried on autonomously replicating plasmids. In each case studied the plasmid-borne gene was transcribed to the same degree as the genomic gene. Nevertheless, the cell maintained a balanced accumulation of ribosomal proteins, using a variety of methods other than transcription. (i) Several ribosomal proteins were synthesized in substantial excess. However, the excess ribosomal protein was rapidly degraded. (ii) The excess mRNA for two of the ribosomal protein genes was translated inefficiently. We provide evidence that this was due to inefficient initiation of translation. (iii) The transcripts derived from two of the ribosomal protein genes were spliced inefficiently, leading to an accumulation of precursor RNA. We present a model which proposes the autogenous regulation of mRNA splicing as a eucaryotic parallel of the autogenous regulation of mRNA translation in procaryotes. Finally, the accumulation of each ribosomal protein was regulated independently. In no instance did the presence of excess copies of the gene for one ribosomal protein affect the synthesis of another ribosomal protein.
Asunto(s)
Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Clonación Molecular , Proteínas Fúngicas/biosíntesis , Biosíntesis de Proteínas , Empalme del ARN , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas Ribosómicas/biosíntesis , Transcripción Genética , Transformación GenéticaRESUMEN
We placed a regulatory sequence derived from the GAL10 locus of Saccharomyces cerevisiae at various distances from the start sites of transcription of two yeast ribosomal protein genes, tcm1 and cyh2. The hybrid ribosomal protein genes were transcribed at wild-type levels in the presence of galactose. In the absence of galactose, the hybrid genes were transcribed either at a reduced level or essentially not at all. Yeast cells which transcribe the ribosomal protein genes at a reduced rate continued to grow, suggesting that enhanced translation of the ribosomal protein mRNA may permit an adequate rate of synthesis of the corresponding protein. Consistent with this suggestion is the finding that preexisting mRNA decayed at a reduced rate when transcription was halted abruptly by removal of galactose. Yeast cells unable to transcribe tcm1 or cyh2 without galactose did not grow. These conditional lethal strains demonstrate that the ribosomal proteins encoded by tcm1 and cyh2 are essential; furthermore, these strains are potentially useful for isolating mutations in the tcm1 and cyh2 proteins affecting their transport, assembly, or function.
Asunto(s)
Genes , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , ADN de Hongos/genética , ADN Recombinante , Galactosa/genética , Regulación de la Expresión Génica , Genes Fúngicos , Ligamiento Genético , Mutación , Regiones Promotoras GenéticasRESUMEN
Mutations in the yeast gene CYH2 can lead to resistance to cycloheximide, an inhibitor of eukaryotic protein synthesis. The gene product of CYH2 is ribosomal protein L29, a component of the 60S ribosomal subunit. We have cloned the wild-type and resistance alleles of CYH2 and determined their nucleotide sequence. Transcription of CYH2 appears to initiate and terminate at multiple sites, as judged by S1 nuclease analysis. The gene is transcribed into an RNA molecule of about 1082 nucleotides, containing an intervening sequence of 510 nucleotides. The splice junction of the intron resides within a codon near the 5' end of the gene. In confirmation of peptide analysis by Stocklein et al. (1) we find that resistance to cycloheximide is due to a transversion mutation resulting in the replacement of a glutamine by glutamic acid in position 37 of L29.
Asunto(s)
Cicloheximida/farmacología , Genes/efectos de los fármacos , Mutación , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Enzimas de Restricción del ADN , Resistencia a Medicamentos , Saccharomyces cerevisiae/efectos de los fármacos , Transcripción GenéticaRESUMEN
Ability to secrete the K1 (or K2) toxin protein and immunity to that toxin [the K1 (or K2) killer trait] are determined by a double-stranded (ds) RNA, called M1 (or M2), whose replication and maintenance depend on at least one of the larger (L) ds RNAs and 29 chromosomal genes, called MAK genes (maintenance of killer). The location of the MAK8 gene near TCM1 (trichodermin resistance) on the yeast map suggested the possible identity of these two genes. Of six independently isolated tcm1 mutants, five were clearly mak-, and the sixth was weakly mak-. In each case, the mak- phenotype and the trichodermin-resistant phenotypes cosegregated in meiosis and showed the expected tight linkage to pet17. The mak- mutations in the trichodermin-resistant strains did not complement mak8-1, indicating that MAK8 and TCM1 are the same gene. The mak8-1 mutation does not make strains resistant to trichodermin, and one tcm1 mutation is only slightly mak-. Whereas tcm1 mutants lose M1 or M2 ds RNA, they do not lose L ds RNA. Because TCM1 codes for ribosomal protein L3 [Fried, H. M. & Warner, J. R. (1981) Proc. Natl. Acad. Sci, USA 78, 238--242], we conclude that ribosomal protein L3 is involved in the replication and maintenance of M ds RNA. Mutations in cyh2 or cry1, producing resistance to cycloheximide and crytopleurine due to mutant ribosomal proteins, do not produce a mak- phenotype. In analogy with bacterial ribosome assembly mutants, yeast low-temperature-sensitive (lts) mutants may have defective ribosomes. We thus examined mutants for an effect on the killer system. An lts5 mutant, unable to grow at 5 degrees C, also has a mak- phenotype (at 30 degrees C) that cosegregates in meiosis with the lts- phenotype. Mutations in seven other lts genes do not result in the mak- phenotype.
Asunto(s)
Genes , ARN Bicatenario/genética , ARN de Hongos/genética , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Mapeo Cromosómico , Mutación , Proteína Ribosomal L3 , Tricodermina/genéticaRESUMEN
The efficient assembly of ribosomes requires a balanced synthesis of ribosomal RNA and each ribosomal protein. In an attempt to establish the mechanisms responsible for such balanced synthesis we have altered the gene dosage for one of the components by introducing into yeast an autonomously replicating plasmid containing the gene tcm1, which codes for ribosomal protein L3. The plasmid is maintained at 5-10 copies per cell by selection for expression of its URA3 gene. The plasmid-containing cells transcribe 7.5 times as much L3 mRNA as control cells, maintain 3.5 times as much L3 mRNA as control cells and synthesize no more than 1.2 times as much L3 protein as control cells. We conclude that the balanced synthesis of ribosomal proteins is maintained by modulating both the efficiency of translation and the lifetime of their mRNAs.
Asunto(s)
Proteínas Ribosómicas/biosíntesis , Levaduras/genética , ADN Recombinante , Regulación de la Expresión Génica , Genes , Plásmidos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/fisiología , Transcripción GenéticaRESUMEN
A cosmid clone bank of yeast DNA has been used to isolate the cycloheximide resistance gene cyh2 of Saccharomyces cerevisiae. A cosmid carrying this gene was identified by cross hybridization to another cloned gene, tsm437. The two genes, which are tightly linked genetically are both present on a 31 kb segment of cloned DNA. The cyh2 gene encodes ribosomal protein L29, a component of the large subunit. Blot hybridization analysis reveals that this gene is present as a single copy in the yeast genome, unlike many other yeast ribosomal protein genes which appear to be duplicated. The cyh2 gene also appears to contain an intervening sequence, a characteristic common to most yeast ribosomal protein genes that have been cloned.
Asunto(s)
Clonación Molecular , Cicloheximida/farmacología , Genes , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Alelos , Enzimas de Restricción del ADN , Farmacorresistencia Microbiana , Hibridación de Ácido Nucleico , Plásmidos , Biosíntesis de Proteínas , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
We have isolated recombinant lambda phage carrying the genes for 14 of the ribosomal proteins of the yeast Saccharomyces cerevisiae. Analysis of these and of the plasmid carrying the gene tcm1, which codes for the ribosomal protein responsible for resistance to trichodermin, demonstrates that in general the genes for ribosomal proteins are unlinked. One exceptional recombinant carries the genes for two ribosomal proteins within a 2-kilobase region. DNA fragments bearing individual ribosomal protein genes were used to probe restriction digests of the yeast genome to determine whether any of the genes were duplicated. Only 3 of 12 of the genes are present unequivocally as a single copy. Similar fragments were used to probe blots of mRNA separated on denaturing agarose gels to determine the size of the mRNA for each protein. In each case, the mRNA is near the minimum size necessary to code for its protein. In certain temperature-sensitive mutants which fail to synthesize functional mRNA for ribosomal protein, Rosbash et al. (Rosbash, M., Harris, P. K. W., Woolford, J., and Teem, J. L. (1981) Cell, 24, 679-686) have demonstrated the accumulation of a larger RNA molecule, homologous to a ribosomal protein gene, that appears to be a transcript which retains an intervening sequence. We find that for 8 of the 11 ribosomal protein genes examined, a larger molecule accumulates in such a mutant strain, suggesting that in general transcripts of ribosomal protein genes may have introns.
Asunto(s)
Genes , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Bacteriófago lambda/genética , Clonación Molecular , Enzimas de Restricción del ADN , ADN Recombinante/metabolismo , Peso Molecular , Plásmidos , ARN Mensajero/genéticaRESUMEN
Yeast cells sensitive to the eukaryotic protein synthesis inhibitor trichodermin have been transformed with autonomously replicating recombinant plasmids carrying DNA fragments of the genome of a trichodermin-resistant yeast strain. After selection for trichodermin-resistant cells, several transformants yielded a plasmid containing a 13.5-kilobase (kb) DNA fragment that encodes the trichodermin resistance gene, tcm1, and the gene for ribosomal protein L3, the largest of the yeast ribosomal proteins. Cells carrying this plasmid are resistant to trichodermin and to the related drug verrucarin A as well as to the unrelated drug anisomycin. This pattern of resistance is similar to that exhibited by strains carrying a chromosomal copy of tcm1. Moreover, polyribosomes prepared from transformed cells are resistant to trichodermin when tested in an in vitro protein synthesis assay. Subcloning of the 13.5-kb DNA fragment revealed that the gene for tcm1 and the gene for protein L3 are contained within a 3.2-kb segment. These results suggest that the gene for trichodermin resistance in yeast specifies ribosomal protein L3.
Asunto(s)
Clonación Molecular , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Sesquiterpenos/farmacología , Tricodermina/farmacología , Anisomicina/farmacología , Cicloheximida/farmacología , ADN de Hongos/genética , Farmacorresistencia Microbiana , Plásmidos , Pirroles/farmacología , Proteína Ribosomal L3 , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Transformación GenéticaRESUMEN
Wild-type and mutant double-stranded RNA (dsRNA) species from the yeast Saccharomyces cerevisiae were studied by electron microscopic heteroduplex mapping to determine the sequence relationships among the different RNA molecules. Three mutant dsRNAs, 1.5, 1.4, and 0.73 kilobase, were found to be derived by the same internal deletion of the wild-type (I83 kilobases) molecule. This deletion includes a wild-type (1.83 kilobases) molecule. This deletion includes a segment of about 200 base pairs that was estimated to be nearly 100% A+U. In addition, the sequences of the two larger mutant RNA species are tandem, direct duplications. One of the duplicated molecules appears to have a second internal deletion that occurred after the duplication. The mutant dsRNAs are functionally similar to the defective interfering virus particles of animal viruses--all of the mutant species prevent the propagation of the wild-type dsRNA when both are present in the same cell. The four dsRNAs share the same sequences at their termini, a finding that may suggest that these sequences are important for the replication of the dsRNAs.