Functional analysis of Saccharomyces cerevisiae ribosomal protein Rpl3p in ribosome synthesis

Ribosome synthesis in eukaryotes requires a multitude of trans-acting factors. These factors act at many steps as the pre-ribosomal particles travel from the nucleolus to the cytoplasm. In contrast to the well-studied trans-acting factors, little is known about the contribution of the ribosomal proteins to ribosome biogenesis. Herein, we have analysed the role of ribosomal protein Rpl3p in 60S ribosomal subunit biogenesis. In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability of early and intermediate pre-ribosomal particles, as evidenced by the low steady-state levels of 27SA₃, 27SB_S and 7S_L/S precursors. Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles. Interestingly, flow cytometry analysis indicates that Rpl3p-depleted cells arrest in the G1 phase. Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented.     

Identification of two SET domain proteins required for methylation of lysine residues in yeast ribosomal protein Rpl42ab

We show that the Saccharomyces cerevisiae ribosomal protein Rpl42ab (the identical product of the RPL42A and RPL42B genes) is monomethylated at Lys-40 and Lys-55. The methylation of Lys-40 is dependent upon the Ybr030w gene product; the methylation of Lys-55 is dependent upon the Set7 gene product. Ybr030w and SET7 genes both encode SET domain containing proteins homologous to known protein lysine methyltransferases, suggesting that their products are the specific enzymes responsible for the monomethylation of the two sites in Rpl42ab. We thus designate Ybr030w as Rkm3 and Set7 as Rkm4. Yeast strains with deletions in both the Ybr030w and SET7 genes produce unmethylated Rpl42ab. A slow growth phenotype was seen for the SET7 deletion strain and the double knock-out when grown in low concentrations of the eukaryotic protein synthesis inhibitor, cycloheximide. These results suggest that modification of Rpl42ab at Lys-55 can fine-tune its structure to avoid inhibition. An intact mass fragmentation approach ("top down mass spectrometry") was used to quantitate the extent of methylation of Rpl42ab. In wild-type strains, it was found that 78% was monomethylated at both Lys-40 and Lys-55 and that 22% was a mixture of species with either Lys-40 or Lys-55 monomethylated. The top down approach was also used to reevaluate the methylation sites of Rpl12ab. We found that the yeast Rpl12ab protein is dimethylated at the N-terminal proline residue, trimethylated at Lys-3 by Rkm2, and monomethylated at Arg-66.     

Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein

The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.     

Structure/function analysis of yeast ribosomal protein L2

Ribosomal protein L2 is a core element of the large subunit that is highly conserved among all three kingdoms. L2 contacts almost every domain of the large subunit rRNA and participates in an intersubunit bridge with the small subunit rRNA. It contains a solvent-accessible globular domain that interfaces with the solvent accessible side of the large subunit that is linked through a bridge to an extension domain that approaches the peptidyltransferase center. Here, screening of randomly generated library of yeast RPL2A alleles identified three translationally defective mutants, which could be grouped into two classes. The V48D and L125Q mutants map to the globular domain. They strongly affect ribosomal A-site associated functions, peptidyltransferase activity and subunit joining. H215Y, located at the tip of the extended domain interacts with Helix 93. This mutant specifically affects peptidyl-tRNA binding and peptidyltransferase activity. Both classes affect rRNA structure far away from the protein in the A-site of the peptidyltransferase center. These findings suggest that defective interactions with Helix 55 and with the Helix 65-66 structure may indicate a certain degree of flexibility in L2 in the neck region between the two other domains, and that this might help to coordinate tRNA-ribosome interactions.     

Mutational analysis of yeast profilin.

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect the roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding in profilin function. A series of truncations was carried out at the C terminus of profilin, a region that has been implicated in actin binding. Removal of the last three amino acids nearly eliminated the ability of profilin to bind polyproline in vitro but had no dramatic in vivo effects. Thus, the extreme C terminus is implicated in polyproline binding, but the physiological relevance of this interaction is called into question. More extensive truncation, of up to eight amino acids, had in vivo effects of increasing severity and resulted in changes in conformation and expression level of the mutant profilins. However, the ability of these mutants to bind actin in vitro was not eliminated, suggesting that this region cannot be solely responsible for actin binding. We also mutagenized a region of profilin that we hypothesized might be involved in PIP2 binding. Alteration of basic amino acids in this region produced mutant profilins that functioned well in vivo. Many of these mutants, however, were unable to suppress the loss of adenylate cyclase-associated protein (Cap/Srv2p [A. Vojtek, B. Haarer, J. Field, J. Gerst, T. D. Pollard, S. S. Brown, and M. Wigler, Cell 66:497-505, 1991]), indicating that a defect could be demonstrated in vivo. In vitro assays demonstrated that the inability to suppress loss of Cap/Srv2p correlated with a defect in the interaction with actin, independently of whether PIP2 binding was reduced. Since our earlier studies of Acanthamoeba profilins suggested the importance of PIP2 binding for suppression, we conclude that both activities are implicated and that an interplay between PIP2 binding and actin binding may be important for profilin function.     

Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlsbergensis

A colony bank of yeast dna obtained by cloning HindIII-generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein mRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticulocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein gene products was further established by electrophoresis on two-dimensional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113 and Y138, have been characterized preliminarily.     

Mutational analysis of the MutH protein from Escherichia coli

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis as PvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) in PvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity both in vivo and in vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHDelta224 and MutHDelta214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).     

The gene and the primary structure of acidic ribosomal protein A0 from yeast Saccharomyces cerevisiae which shows partial homology to bacterial ribosomal protein L10

Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes. By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1,614 nucleotides including about 500 nucleotides in the 5'-flanking region. The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region. The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD. In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA. On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue.     

Altered ribosomal protein S11 from the SUP46 suppressor of yeast

The dominant suppressor SUP46 of the yeast Saccharomyces cerevisiae was shown to act on a wide range of mutations (preceding paper by Ono et al., 1981). Masurekar et al. (1981) demonstrated that ribosomes from the SUP46 strain make an abnormally high rate of errors in a cell-free translation system. These findings indicated that SUP46 suppression was the result of abnormal ribosomes misreading mutant codons. We have used two-dimensional polyacrylamide gel electrophoresis to show that the S11 protein from the 40 S ribosomal subunit has an altered electrophoretic mobility. Thus the gene product of the SUP46 locus is either the S11 ribosomal protein or an enzyme that modifies the S11 protein. These results demonstrate that the altered S11 protein is responsible for the suppression by misreading.     

Mutational analysis of subunit G (Vma10p) of the yeast vacuolar H+-ATPase

The G subunit of V-ATPases is a soluble subunit that shows homology with the b subunit of F-ATPases and may be part of the "stator" stalk connecting the peripheral V(1) and membrane V(0) sectors. When the N-terminal half of the G subunit is modeled as an alpha helix, most of the conserved residues fall on one face of the helix (Hunt, I. E., and Bowman, B. J. (1997) J. Bioenerg. Biomembr. 29, 533-540). We probed the function of this region by site-directed mutagenesis of the yeast VMA10 gene. Stable G subunits were produced in the presence of Y46A and K55A mutations, but subunit E was destabilized, resulting in loss of the V-ATPase assembly. Mutations E14A and K50A allowed wild-type growth and assembly of V-ATPase complexes, but the complexes formed were unstable. Mutations R25A and R25L stabilized V-ATPase complexes relative to wild-type and partially inhibited disassembly of V(1) from V(0) in response to glucose deprivation even though the mutant enzymes were fully active. A 2-amino acid deletion in the middle of the predicted N-terminal helix (DeltaQ29D30) allowed assembly of a functional V-ATPase. The results indicate that, although the N-terminal half of the G subunit is essential for V-ATPase activity, either this region is not a rigid helix or the presence of a continuous, conserved face of the helix is not essential.     

Mutational analysis of the fission yeast p34cdc2 protein kinase gene

Summary The p34^cdc2 protein serine-threonine kinase plays an essential role in the life cycle of fission yeast, being required for both the G1-S and G2-M transitions during mitotic growth, and also for the second meiotic nuclear division. Functional homologues of p34^cdc2 (each ca. 60 % identical to the fission yeast prototype) have been isolated from organisms as diverse as humans, insects and plants, and there is now considerable evidence supporting the view that fundamental aspects of the cell cycle controls uncovered in fission yeast will prove to be conserved in all eukaryotes. By comparing the amino acid sequences of fission yeast p34^cdc2 with its higher eukaryotic counterparts it is possible to identify conserved residues that are likely to be centrally important for p34^cdc2 function. Here the effects are described of mutating a number of these conserved residues. Twenty-three new mutant alleles have been constructed and tested. We show that replacing cysteine 67 with trypthophan renders the resulting mutant protein p80^cdc25-independent (while neither leucine, isoleucine nor valine has this effect) and that several of the amino acids within the highly conserved PSTAIRE region are not absolutely required for p34^cdc2 function. Five acidic amino acids have also been mutated within p34^cdc2, which are invariant across the eukaryotic protein kinase family. Acid-to-base mutations at three of these residues resulted in a dominant-negative, cell cycle arrest phenotype while similar mutations at the other two simply abolished p34^cdc2 protein function. The results are discussed with reference to the predicted tertiary structure of the p34^cdc2 enzyme.     

Interaction of Nascent Chains with the Ribosomal Tunnel Proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae

As translation proceeds, nascent polypeptides pass through an exit tunnel that traverses the large ribosomal subunit. Three ribosomal proteins, termed Rpl4, Rpl17, and Rpl39 expose domains to the interior of the exit tunnel of eukaryotic ribosomes. Here we generated ribosome-bound nascent chains in a homologous yeast translation system to analyze contacts between the tunnel proteins and nascent chains. As model proteins we employed Dap2, which contains a hydrophobic signal anchor (SA) segment, and the chimera Dap2α, in which the SA was replaced with a hydrophilic segment, with the propensity to form an α-helix. Employing a newly developed FLAG exposure assay, we find that the nascent SA segment but not the hydrophilic segment adopted a stable, α-helical structure within the tunnel when the most C-terminal SA residue was separated by 14 residues from the peptidyl transferase center. Using UV cross-linking, antibodies specifically recognizing Rpl17 or Rpl39, and a His₆-tagged version of Rpl4, we established that all three tunnel proteins of yeast contact the SA, whereas only Rpl4 and Rpl39 also contact the hydrophilic segment. Consistent with the localization of the tunnel exposed domains of Rpl17 and Rpl39, the SA was in contact with Rpl17 in the middle region and with Rpl39 in the exit region of the tunnel. In contrast, Rpl4 was in contact with nascent chain residues throughout the ribosomal tunnel.     

酵母核糖体蛋白基因组合转录调控位点统计分析

真核基因的转录调控是后基因组时代研究的主要问题之一,其基础是认识DNA上转录因子结合位点(模体)及分布状况。基于马尔可夫链模型对酵母核糖体蛋白基因上游启动子序列中模体出现次数进行统计,利用Z-score统计量抽提出过表达和低表达的模体,其中95%的模体与实验得到的转录因子结合位点相符合。然后将抽提出的模体两两配对,通过与背景序列比较,找出酵母核糖体蛋白基因中出现概率及距离分布均具有统计显著性的模体对,这些非随机出现的模体对具有潜在的组合转录调控功能,其中一些模体对的组合调控作用已有实验支持。对提取出的模体对在序列中的位置分布进行分析,发现近94%的模体对位于转录起始位点上游,超过半数的模体对两模体之间的最短距离在0～100bp之间,距离小于30bp的模体对接近30%,这样的短距离间隔有利于两模体的相同作用。这些结果将有助于对酵母核糖体蛋白基因转录调控机制的深入认识。     

Mutational analysis of the -10 region from the Mycobacterium tuberculosis lipF promoter

The promoter driving expression of the Mycobacterium tuberculosis gene lipF (Rv3487c) had previously been identified as being upregulated by acidic stress. Subsequently a 59 base pair (bp) acid inducible minimal promoter region was isolated in which a putative -10 region was identified. In this study we use mutational analysis to investigate the -10 region of the lipF promoter. Mutations within this region lead to a dramatic decrease of promoter activity, while a mutation outside of this region does not affect promoter activity.     

Structural comparison of yeast ribosomal protein genes.

The primary structure of the genes encoding the yeast ribosomal proteins L17a and L25 was determined, as well as the positions of the 5'- and 3'-termini of the corresponding mRNAs. Comparison of the gene sequences to those obtained for various other yeast ribosomal protein genes revealed several similarities. In all split genes the intron is located near the 5'-side of the amino acid coding region. Among the introns a clear pattern of sequence conservation can be observed. In particular the intron-exon boundaries and a region close to the 3'-splice site show sequence homology. Conserved sequences were also found in the leader and trailer regions of the ribosomal protein mRNAs. The 5'-flanking regions of the yeast ribosomal protein genes appeared to contain sequence elements that many but not all ribosomal protein genes have in common, and therefore may be implicated in the coordinate expression of these genes. The amino acid coding sequences of the ribosomal protein genes show a biased codon usage. Like most yeast ribosomal protein molecules, L17a and L25 are particularly basic at their N-terminus.