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.     

Characterization of two types of yeast ribosomal DNA genes.

The intragenic organization of ribosomal DNA from a diploid strain of Saccharomyces cerevisiae was analyzed by using recombinant DNA molecules constructed in vitro. Restriction analysis of the yeast ribosomal DNA with the EcoRI restriction enzyme indicated that eight restriction fragments were present in the ribosomal DNA of this strain: X' (1.87 X 10(6) daltons), A (1.77 X 10(6) daltons), B (1.48 X 10(6) daltons), C (1.22 X 10(6) daltons), D (0.39 X 10(6) daltons), E (0.36 X 10(6) daltons), F (0.22 X 10(6) daltons), and G (0.17 X 10(6) daltons). These fragments were distributed between two different types of ribosomal DNA genes, which had the restriction maps: (formula: see text) in which the underlined region shows the repeating unit. The diploid yeast strain contained approximately equal amounts of each of these two types of genes. The analysis of the recombinant DNA molecules also indicated that the yeast ribosomal genes are homogeneous and extensively clustered.     

The yeast ribosomal protein S7 and its genes.

Ribosomal protein S7 of Saccharomyces cerevisiae is encoded by two genes RPS7A and RPS7B. The sequence of each copy was determined; their coding regions differ in only 14 nucleotides, none of which leads to changes in the amino acid sequence. The predicted protein consists of 261 amino acids, making it the largest protein of the 40 S ribosomal subunit. It is highly basic near the NH2 terminus, as are most ribosomal proteins. Protein S7 is homologous to both human and rat ribosomal protein S4. RPS7A and RPS7B contain introns of 257 and 269 nucleotides, respectively, located 11 nucleotides beyond the initiator AUG. The splicing of the introns is efficient. Either RPS7A or RPS7B will support growth. However, deletion of both genes is lethal. RPS7A maps distal to CDC11 on chromosome X, and RPS7B maps distal to CUP1 on chromosome VIII.     

The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome

Eukaryotic ribosomes are made of two components, four ribosomal RNAs, and approximately 80 ribosomal proteins (r-proteins). The exact number of r-proteins and r-protein genes in higher plants is not known. The strong conservation in eukaryotic r-protein primary sequence allowed us to use the well-characterized rat (Rattus norvegicus) r-protein set to identify orthologues on the five haploid chromosomes of Arabidopsis. By use of the numerous expressed sequence tag (EST) accessions and the complete genomic sequence of this species, we identified 249 genes (including some pseudogenes) corresponding to 80 (32 small subunit and 48 large subunit) cytoplasmic r-protein types. None of the r-protein genes are single copy and most are encoded by three or four expressed genes, indicative of the internal duplication of the Arabidopsis genome. The r-proteins are distributed throughout the genome. Inspection of genes in the vicinity of r-protein gene family members confirms extensive duplications of large chromosome fragments and sheds light on the evolutionary history of the Arabidopsis genome. Examination of large duplicated regions indicated that a significant fraction of the r-protein genes have been either lost from one of the duplicated fragments or inserted after the initial duplication event. Only 52 r-protein genes lack a matching EST accession, and 19 of these contain incomplete open reading frames, confirming that most genes are expressed. Assessment of cognate EST numbers suggests that r-protein gene family members are differentially expressed.     

The organization of ribosomal genes in vertebrates

The organization of the repeat unit of the ribosomal genes has been determined in 15 different species of vertebrates. The EcoRI and BamHI restriction maps of the rDNA from single individuals of different species of fishes, amphibians, and reptiles have been analysed. Two rDNA clones from Xenopus laevis (representing one complete repeat unit) were used as probes in Southern blots to detect restriction fragments containing ribosomal genes. The results obtained indicate that the transcribed regions are highly conserved in length and sequence inside the same zoological class. These regions are less conserved when species from different classes are compared but a general trend has been observed. In contrast, the length and sequence of the spacer regions are very variable, even within the same zoological class. Different types of heterogeneity have been observed; examples range from a single type of ribosomal repeat unit within a species to the absence of any detectable regular tandem array of units.     

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.     

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.     

Cloning and analysis of the nuclear genes for two mitochondrial ribosomal proteins in yeast

Summary Two mitochondrial ribosomal proteins of yeast (Saccharomyces cerevisiae) were purified and their N-terminal amino acid sequences determined. The sequence data were used for the synthesis of oligonucleotide probes to clone the corresponding genes. Thus, the genes for two proteins, termed YMR-31 and YMR-44, were cloned and their nucleotide sequences determined. From the nucleotide sequence data, the coding region of the gene for protein YMR-31 was found to be composed of 369 nucleotide pairs. Comparison of the amino acid sequence of protein YMR-31 and the one deduced from the nucleotide sequence of its gene suggests that it contains an octapeptide leader sequence. The calculated molecular weight of protein YMR-31 without the leader sequence is 12792 dalton. The gene for protein YMR-44 was found to contain a 147 bp intron which contains two sequences conserved among yeast introns. The length of the two exons flanking the intron totals 294 nucleotide pairs which can encode a protein with a calculated molecular weight of 11476 dalton. The gene for protein YMR-31 is located on chromosome VI, while the gene for protein YMR-44 is located on either chromosome XIII or XVI.     

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。     

Similar organization of beta,beta'-subunit RNA-polymerase genes and adjacent ribosomal protein genes in Enterobacteriaceae and Pseudomonas putida

The genes coding for the RNA-polymerase beta,beta'-subunits and adjacent ribosomal protein genes in Escherichia coli, Salmonella typhimurium, Shigella flexneri, Serratia marcescens, Proteus mirabilis and Pseudomonas putida are compared by the Southern hybridization procedure. In all the species studied close clustering of the genes rplKAJL and rpoBC is demonstrated. Preliminary physical maps for these genes in S. typhimurium, S. flexneri, S. marcescens and P. mirabilis are proposed. Rifampicin is shown to stimulate the beta,beta'-subunit synthesis in all the species studied, suggesting the existence of attenuators localized in front of the rpoBC genes. The similar arrangement of the genes rplKAJLrpoBC in a number of bacterial species is proposed to be due to common mechanisms of their coordinate expression.     

Introns within ribosomal protein genes regulate the production and function of yeast ribosomes

In budding yeast, the most abundantly spliced pre-mRNAs encode ribosomal proteins (RPs). To investigate the contribution of splicing to ribosome production and function, we systematically eliminated introns from all RP genes to evaluate their impact on RNA expression, pre-rRNA processing, cell growth, and response to stress. The majority of introns were required for optimal cell fitness or growth under stress. Most introns are found in duplicated RP genes, and surprisingly, in the majority of cases, deleting the intron from one gene copy affected the expression of the other in a nonreciprocal manner. Consistently, 70% of all duplicated genes were asymmetrically expressed, and both introns and gene deletions displayed copy-specific phenotypic effects. Together, our results indicate that splicing in yeast RP genes mediates intergene regulation and implicate the expression ratio of duplicated RP genes in modulating ribosome function.     

Functional analysis of a duplicated linked pair of ribosomal protein genes in Saccharomyces cerevisiae.

Ribosomal protein genes RP28 and S16A (RP55) are closely linked. Another set of this pair of genes exists in the genome (copy 2), genetically unlinked to copy 1. By using gene replacement techniques, we have shown that RP28 from copy 1 is required for vegetative growth and that the cells need S16A from copy 2 to achieve maximum growth rate.