Independent genes coding for three acidic proteins of the large ribosomal subunit from Saccharomyces cerevisiae

The yeast ribosome contains three acidic proteins, L44, L44', and L45, closely related from a structural point of view, that seem to play a functional role similar to that of proteins L7 and L12 in the bacterial ribosome. By screening a cDNA bank in lambda gt11 with specific polyclonal and monoclonal antibodies, recombinant phages expressing each one of the acidic proteins have been cloned. A unique copy of each gene is detected using the phage cDNA inserts as probes in nitrocellulose blots of yeast DNA digested with different restriction enzymes. The inserts were subcloned in the plasmid pUC19, and their physical maps and nucleotide sequences were determined. By using the cDNA inserts as probes in genomic DNA banks, DNA fragments carrying the acidic protein genes have been cloned, characterized, and sequenced. The results conclusively show that the three yeast acidic proteins are coded by independent genes and are not the result of a post-translational modification of the product of a unique gene, as in bacteria. Like most ribosomal protein genes, the gene for protein L44' has an intron and two upstream stimulatory boxes (UASrpg) fitting closely to the consensus sequence. The genes coding for proteins L44 and L45 lack introns and seem also exceptional in other characteristics of their sequences. Proteins L44 and L45 have amino acid sequences with about 80% similarity. Protein L44' is only 63% similar to the other two polypeptides. The three proteins have highly conserved carboxyl termini comprising the last 30 amino acids, and the first 10 amino acids of L44 and L45 are identical. The results cast doubts about the possibility of a similar role for the different acidic ribosomal proteins.     

Stable binding of the eukaryotic acidic phosphoproteins to the ribosome is not an absolute requirement for in vivo protein synthesis.

The genes encoding the four acidic ribosomal phosphoproteins have been inactivated in Saccharomyces cerevisae by recombination with truncated genes carrying different genetic markers. By crossing single haploid disruptants, strains harboring two simultaneously inactivated acidic protein genes were constructed. None of the six possible double disruptions was lethal, but the simultaneous inactivation of either YP1 alpha and YP1 beta(L44') or YP2 alpha(L44) and YP2 beta(L45) caused an important decrease in the cell growth rate. Ribosomes isolated from these slow-growing strains did not contain acidic proteins, not even the two polypeptides whose genes were still intact, although these proteins were present in the cell extracts and they seem to be able to form high-molecular weight protein complexes. Transformation of a slow-growing double transformant with a plasmid containing one of the disrupted genes restored the presence of the acidic proteins in the ribosomes and normal growth rates. The particles of the slow-growing strains were active in an in vitro amino acid polymerizing system, although their activity could be stimulated by the exogenous addition of the missing proteins. These results indicate that in the absence of either YP1 alpha and YP1 beta(L44') or YP2 alpha (L44) and YP2 beta(L45), the remaining acidic proteins are unable to interact with the ribosome in a stable manner, but that a strong interaction of these ribosomal components with the particle is not an absolute requirement for in vivo and in vitro protein synthesis.     

Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.     

Characterization of the yeast acidic ribosomal phosphoproteins using monoclonal antibodies. Proteins L44/L45 and L44' have different functional roles.

In order to characterize the acidic ribosomal proteins immunologically and functionally, a battery of monoclonal antibodies specific for L44, L44' and L45, the three acidic proteins detected in Saccharomyces cerevisiae, were obtained. Eight monoclonal antibodies were obtained specific for L45, three for L44' and one for L44. In addition, two mAbs recognizing only the phosphorylated forms of the three proteins were obtained. The specific immunogenic determinants are located in the middle region of the protein structure and are differently exposed in the ribosomal surface. The common determinants are present in the carboxyl end of the three proteins. An estimation of the acidic proteins by ELISA indicated that, in contrast to L44 and L45, L44' is practically absent from the cell supernatant; this suggests that protein L44' does not intervene in the exchange that has been shown to take place between the acidic proteins in the ribosome and in the cytoplasmic pool. It has also been found that, while IgGs specific for L44 and L45 do not inhibit the ribosome activity, the anti-L44' effectively blocks the polymerizing activity of the particles. These results show for the first time that the different eukaryotic acidic ribosomal proteins play a different functional role.     

Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves translational accuracy, while mutants lacking both remain viable

Four mutant strains from Saccharomyces cerevisiae were used to study ribosome structure and function. They included a strain carrying deletions of the two genes encoding ribosomal protein L24, a strain carrying a mutation spb2 in the gene for ribosomal protein L39, a strain carrying a deletion of the gene for L39, and a mutant lacking both L24 and L39. The mutant lacking only L24 showed just 25% of the normal polyphenylalanine-synthesizing activity followed by a decrease in P-site binding, suggesting the possibility that protein L24 is involved in the kinetics of translation. Each of the two L39 mutants displayed a 4-fold increase of their error frequencies over the wild type. This was accompanied by a substantial increase in A-site binding, typical of error-prone mutants. The absence of L39 also increased sensitivity to paromomycin, decreased the ribosomal subunit ratio, and caused a cold-sensitive phenotype. Mutant cells lacking both ribosomal proteins remained viable. Their ribosomes showed reduced initial rates caused by the absence of L24 but a normal extent of polyphenylalanine synthesis and a substantial in vivo reduction in the amount of 80S ribosomes compared to wild type. Moreover, this mutant displayed decreased translational accuracy, hypersensitivity to the antibiotic paromomycin, and a cold-sensitive phenotype, all caused mainly by the deletion of L39. Protein L39 is the first protein of the 60S ribosomal subunit implicated in translational accuracy.     

Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.     

Ribosomes with large synthetic N-terminal extensions of protein S15 are active in vivo

The genes for ribosomal proteins S4, S13 or S15 were fused with the gene for staphylococcal protein A, or derivatives thereof (2A'-7A'). The gene fusions were introduced into Escherichia coli strains, mutated in the corresponding ribosomal protein gene, by transformation. These mutated ribosomal proteins cause a phenotype that can be complemented. Thus, the phenotype of the transformants was tested and the ribosomal proteins were analyzed. The S4 N-terminal fusion protein severely disturbed growth of both the mutant and the wild-type strains. The S13 C-terminal fusion protein was proteolyzed close to the fusion point, giving a ribosomal protein moiety that could assemble into the ribosome normally. S15 N-terminal fusion proteins complemented a cold-sensitive strain lacking protein S15 in its ribosomes. These fused proteins were assembled into active ribosomes. The position of S15 in the 30S ribosomal subunit is well known. Therefore, in structural studies of the ribosome in vivo, the S15 fusion proteins can be used as a physical reporter for S15.     

Importance of the 5 S rRNA-binding ribosomal proteins for cell viability and translation in Escherichia coli

A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild-type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.     

Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis.

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis.     

Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.     

Isolation and structural analysis of ribosomal protein genes in Xenopus laevis. Homology between sequences present in the gene and in several different messenger RNAs

The ribosomal protein genes are present in two to four copies per haploid genome of Xenopus laevis. Using cloned complementary DNA probes, we have isolated, from a genomic library of X. laevis, several clones containing genes for two different ribosomal proteins (L1 and L14). These genes contain intervening sequences. In the case of the L1 gene, the exons are 100 to 200 base-pairs long and the introns, on average, 400 base-pairs. Along the genomic fragments, two different classes of repetitive DNA are present: highly and middle repetitive DNA. Both are evolutionarily unstable as shown by hybridization to Xenopus tropicalis DNA. Several introns of the gene coding for protein L1 contain middle repetitive sequences. Hybridization and hybrid-released translation experiments have shown that sequences inside the two genes hybridize to several poly(A) messenger RNAs. Some of the products encoded by these mRNA have electrophoretic properties of ribosomal proteins.     

The 26S rRNA binding ribosomal protein equivalent to bacterial protein L11 is encoded by unspliced duplicated genes in Saccharomyces cerevisiae.

Transformant phages expressing L15, a yeast ribosomal protein which binds to 26S rRNA and interacts with the acidic ribosomal proteins, were isolated by screening a yeast cDNA expression library in lambda gt11 with specific monoclonal antibodies. Using yeast DNA HindIII fragments that hybridize with the cDNA insert from the L15-expressing clones, minilibraries were prepared in pUC18, which were afterward screened with the same cDNA probe. In this way, plasmids carrying two different types of genomic DNA inserts were obtained. The inserts were subcloned and sequenced and we found a similar coding sequence in both cases flanked by 5' and 3' regions with very low homology. Sequences homologous to the consensus TUF-binding UAS boxes are present in the 5' flanking regions of both genes. Southern analysis revealed the presence of two copies of the L15 gene in the Saccharomyces cerevisiae genome, which are located in different chromosomes. The encoded amino acid sequence corresponds, as expected, to protein L15 and shows a high similarity to bacterial ribosomal protein L11.     

Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol. To improve characteristics of xylose fermentation, the recombinant strain Delta xyl1 Delta xyl2-ADelta xyl2-B, with deletions of genes encoding first enzymes of xylose utilization (NAD(P)H-dependent xylose reductase and NAD-dependent xylitol dehydrogenases, respectively), was constructed and used as a recipient for co-overexpression of the Escherichia coli xylA gene coding for xylose isomerase and endogenous XYL3 gene coding for xylulokinase. The expression of both genes was driven by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter. Xylose isomerase activities of obtained transformants amounted to approximately 80% of that of the bacterial host strain. Xylulokinase activities of the transformants increased twofold when compared with the parental strain. The recombinant strains displayed improved ethanol production during the fermentation of xylose.     

Phosphoprotein phosphatase from yeast and its ribosomal substrate

Phosphoprotein phosphatase was isolated from yeast postribosomal supernatant and partly characterized. The enzyme preferentially dephosphorylated phospho-casein and acidic ribosomal proteins L44 and L45, the eukaryotic analogues of bacterial proteins L7 and L12. The evidence suggests that this enzyme is not a catalytic subunit of the multifunctional phosphoprotein phosphatase present in most eukaryotic organisms.     

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.