Showing papers on "Ribosomal protein published in 2001"

Crystal structure of the ribosome at 5.5 A resolution

Abstract: We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.

Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria

Abstract: Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg+2 ions for the binding of some drugs. This structural analysis should facilitate rational drug design.

The kink-turn: a new RNA secondary structure motif.

Abstract: Analysis of the Haloarcula marismortui large ribosomal subunit has revealed a common RNA structure that we call the kink-turn, or K-turn. The six K-turns in H.marismortui 23S rRNA superimpose with an r.m.s.d. of 1.7 A. There are two K-turns in the structure of Thermus thermophilus 16S rRNA, and the structures of U4 snRNA and L30e mRNA fragments form K-turns. The structure has a kink in the phosphodiester backbone that causes a sharp turn in the RNA helix. Its asymmetric internal loop is flanked by C-G base pairs on one side and sheared G-A base pairs on the other, with an A-minor interaction between these two helical stems. A derived consensus secondary structure for the K-turn includes 10 consensus nucleotides out of 15, and predicts its presence in the 5'-UTR of L10 mRNA, helix 78 in Escherichia coli 23S rRNA and human RNase MRP. Five K-turns in 23S rRNA interact with nine proteins. While the observed K-turns interact with proteins of unrelated structures in different ways, they interact with L7Ae and two homologous proteins in the same way.

Macrolide Resistance Conferred by Base Substitutions in 23S rRNA

Abstract: Resistance to all major groups of antibiotics has arisen hand in hand with their extensive use in medicine and animal husbandry, and macrolide antibiotics are no exception. The therapeutic utility of macrolides has been severely compromised by the emergence of drug resistance in many pathogenic bacteria. The molecular mechanisms by which bacteria become resistant are manifold, but in general these can be collectively characterized as involving either drug efflux, drug inactivation, or alterations in the drug target site. The target site for macrolides is the large (50S) subunit of the bacterial ribosome. Many cases of macrolide resistance in clinical strains can be linked to alteration of specific nucleotides in 23S rRNA within the large ribosomal subunit. Macrolides are natural polyketide products of secondary metabolism in many actinomycete species (51, 140). Clinically useful macrolides consist of a 14-, 15-, or 16-member lactone ring (Table ​(Table1)1) that is generally substituted with two or more neutral and/or amino sugars (16). The structures of the 14- and 16-member-ring macrolides erythromycin and tylosin and of some semisynthetic erythromycin derivatives are shown in Fig. ​Fig.1.1. The inhibitory action of erythromycin, and probably that of the other 14-member-ring macrolides, is effected at the early stages of protein synthesis when the drug blocks the growth of the nascent peptide chain (7, 140), presumably causing premature dissociation of the peptidyl-tRNA from the ribosome (85). The antimicrobial action of these drugs is compounded by their inhibition of the assembly of new large ribosomal subunits, which leads to gradual depletion of functional ribosomes in the cell (23). The mode of action of the 16-member-ring macrolides is less well characterized, although it is clear that they bind to the same region of the large subunit as the 14-member-ring macrolides and inhibit peptide bond formation in a more direct manner (reviewed in reference 140). TABLE 1 Macrolide antibiotics and their derivatives discussed in this review FIG. 1 Selected clinically important macrolide antibiotics and their derivatives. Two naturally occurring macrolides are shown: erythromycin A, which was the first therapeutic macrolide and possesses a 14-member ring, and tylosin, a 16-member-ring macrolide ... Shortly after the introduction of erythromycin in therapy in the 1950s, resistance to the drug was observed in bacterial pathogens (reviewed in reference 76). More disquieting was the observation that erythromycin-resistant strains were cross-resistant not only to all other macrolides but also to the chemically unrelated lincosamide and streptogramin B drugs. This phenomenon was first observed in Staphylococcus aureus and came to be termed the macrolide-lincosamide-streptogramin B (MLSB) antibiotic resistance phenotype. In these S. aureus strains, MLSB resistance can be induced by exposure to low concentrations of erythromycin (151), which leads to expression of a methyltransferase enzyme (ErmC). ErmC specifically methylates 23S rRNA (74) at the N-6 position of adenosine 2058 (A2058) (Escherichia coli numbering) (121), which is a pivotal nucleotide for the binding of MLSB antibiotics (see below). Subsequently, several dozen erm methyltransferase genes have been identified. Many of these are constitutively expressed, and their products all presumably methylate A2058. A new nomenclature system has recently been proposed for the different erm genes, which clarifies their phylogenetic relatedness (105). For a comprehensive account of the action of Erm methyltransferases, see the review by Weisblum (149). Since the discovery of erm genes, another means of resistance involving alteration of rRNA structure has been identified. Under laboratory conditions, single base substitutions introduced into rRNA were shown to confer macrolide resistance. This form of resistance was first observed in the single rRNA (rrn) operon of yeast mitochondria, which was mutated at position A2058 in the large-subunit rRNA (123). Shortly afterwards, similar phenotypes were obtained in E. coli by expression of mutant rrn alleles from multiple-copy plasmids (see, e.g., references 120 and 143). About 6 years ago, reports of rRNA mutations conferring macrolide resistance in clinical pathogens began to appear in the literature. While it is conceptually gratifying to establish that the mutations appearing in pathogens are identical to those previously isolated in laboratory strains, the clinical implications of this are quite disturbing. The 23S rRNA mutations reported so far to cause macrolide resistance are shown in Table ​Table2.2. Generally, pathogenic species that develop macrolide resistance through mutations at A2058 (or neighboring nucleotides) possess only one or two rrn operons, such as in the case of Helicobacter pylori and Mycobacterium species. Resistance in bacteria with multiple rrn operons, such as Enterococcus, Streptococcus, and Staphylococcus species, is generally conferred by Erm methylation of A2058 (Table ​(Table3)3) or by efflux (see e.g., references 70 and 110). However, there are cases of macrolide resistance by drug inactivation (reviewed in reference 150), and there are recent reports of macrolide resistance in Streptococcus pneumoniae strains conferred by mutations in ribosomal proteins L4 and L22 and in rRNA (129; P. Appelbaum, personal communication). Macrolide and ketolide resistance is additionally conferred in E. coli by the expression of small, specific peptides (134), although the level of resistance is probably too low to be a problem in the treatment of clinical strains. TABLE 2 23S rRNA mutations reported to confer macrolide resistance TABLE 3 Macrolide resistance mechanisms found in some pathogens and their numbers of rRNA operons In the following sections of this review, we first look at the current state of knowledge of the bacterial ribosome target site for macrolide antibiotics. A detailed model of a drug target site is a prerequisite for understanding the molecular mechanisms of drug binding and drug resistance and for rational design of new drugs. Our present state of knowledge, although far from being complete, supports the view that the macrolide target site is highly conserved within the ribosomes of all bacteria. We then direct attention to the pathogens, and in particular to H. pylori, that have been shown to attain resistance by rRNA mutation, and we consider the possibility of this form of resistance emerging in other pathogens. Finally, some suggestions are made regarding how future macrolide derivatives might be best equipped to combat bacteria with resistant rRNAs.

Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3

Abstract: The small ribosomal subunit is responsible for the decoding of genetic information and plays a key role in the initiation of protein synthesis. We analyzed by X-ray crystallography the structures of three different complexes of the small ribosomal subunit of Thermus thermophilus with the A-site inhibitor tetracycline, the universal initiation inhibitor edeine and the C-terminal domain of the translation initiation factor IF3. The crystal structure analysis of the complex with tetracycline revealed the functionally important site responsible for the blockage of the A-site. Five additional tetracycline sites resolve most of the controversial biochemical data on the location of tetracycline. The interaction of edeine with the small subunit indicates its role in inhibiting initiation and shows its involvement with P-site tRNA. The location of the C-terminal domain of IF3, at the solvent side of the platform, sheds light on the formation of the initiation complex, and implies that the anti-association activity of IF3 is due to its influence on the conformational dynamics of the small ribosomal subunit.

RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: A negative feedback circuit

Abstract: The RPN4 (SON1, UFD5) protein of the yeast Saccharomyces cerevisiae is required for normal levels of intracellular proteolysis. RPN4 is a transcriptional activator of genes encoding proteasomal subunits. Here we show that RPN4 is required for normal levels of these subunits. Further, we demonstrate that RPN4 is extremely short-lived (t(1/2) approximate to 2 min), that it directly interacts with RPN2, a subunit of the 26S proteasome, and that rpn4 Delta cells are perturbed in their cell cycle. The degradation signal of RPN4 was mapped to its N-terminal region, outside the transcription-activation domains of RPN4. The ability of RPN4 to augment the synthesis of proteasomal subunits while being metabolically unstable yields a negative feedback circuit in which the same protein up-regulates the proteasome production and is destroyed by the assembled active proteasome.

N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis.

Abstract: The myc oncogenes are frequently activated in human tumors, but there is no comprehensive insight into the target genes and downstream cellular pathways of these transcription factors. We applied serial analysis of gene expression (SAGE) to identify targets of N-myc in neuroblastomas. Analysis of 42,000 mRNA transcript tags in SAGE libraries of N-myc- transfected and control neuroblastoma cells revealed 114 up-regulated genes. The majority of these genes have a role in ribosome assembly and activity. Northern blot analysis confirmed up-regulation of all tested transcripts. Induction was complete within 4 h after N-myc expression. The large majority of the ribosomal proteins were induced, as well as genes controlling rRNA maturation. Cellular rRNA content was 45% induced. SAGE libraries and northern blot analysis confirmed up-regulation of many of these genes in N-myc-amplified neuroblastomas. As N-myc can functionally replace c-myc, we analyzed whether N-myc targets were induced by c-myc as well. Approximately 40% of these N-myc targets were up-regulated in a c-myc-transfected melanoma cell line. These data suggest that myc genes function as major regulators of the protein synthesis machinery.

Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E. coli.

Abstract: Inorganic polyphosphate (polyP), a polymer of hundreds of phosphate (Pi) residues, accumulates in Escherichia coli in response to stresses, including amino acid starvation. Here we show that the adenosine 5'-triphosphate-dependent protease Lon formed a complex with polyP and degraded most of the ribosomal proteins, including S2, L9, and L13. Purified S2 also bound to polyP and formed a complex with Lon in the presence of polyP. Thus, polyP may promote ribosomal protein degradation by the Lon protease, thereby supplying the amino acids needed to respond to starvation.

Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p.

Abstract: Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.

Codon usage and tRNA genes in eukaryotes: correlation of codon usage diversity with translation efficiency and with CG-dinucleotide usage as assessed by multivariate analysis.

Abstract: The species-specific diversity of codon usage in five eukaryotes (Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and Homo sapiens) was investigated with principal component analysis. Optimal codons for translation were predicted on the basis of tRNA-gene copy numbers. Highly expressed genes, such as those encoding ribosomal proteins and histones in S. pombe, C. elegans, and D. melanogaster, have biased patterns of codon usage which have been observed in a wide range of unicellular organisms. In S. pombe and C. elegans, codons contributing positively to the principal component with the largest variance (Z1-parameter) corresponded to the optimal codons which were predicted on the basis of tRNA gene numbers. In D. melanogaster, this correlation was less evident, and the codons contributing positively to the Z1-parameter corresponded primarily to codons with a C or G in the codon third position. In X. laevis and H. sapiens, codon usage in the genes encoding ribosomal proteins and histones was not significantly biased, suggesting that the primary factor influencing codon-usage diversity in these species is not translation efficiency. Codon-usage diversity in these species is known to reflect primarily isochore structures. In the present study, the second additional factor was explained by the level of use of codons containing CG-dinucleotides, and this is discussed with respect to transcription regulation via methylation of CG-dinucleotides, which is observed in mammalian genomes.

The Organization of Cytoplasmic Ribosomal Protein Genes in the Arabidopsis Genome

Abstract: 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.

KH domain: one motif, two folds

Abstract: The K homology (KH) module is a widespread RNA-binding motif that has been detected by sequence similarity searches in such proteins as heterogeneous nuclear ribonucleoprotein K (hnRNP K) and ribosomal protein S3. Analysis of spatial structures of KH domains in hnRNP K and S3 reveals that they are topologically dissimilar and thus belong to different protein folds. Thus KH motif proteins provide a rare example of protein domains that share significant sequence similarity in the motif regions but possess globally distinct structures. The two distinct topologies might have arisen from an ancestral KH motif protein by N- and C-terminal extensions, or one of the existing topologies may have evolved from the other by extension, displacement and deletion. C-terminal extension (deletion) requires β-sheet rearrangement through the insertion (removal) of a β-strand in a manner similar to that observed in serine protease inhibitors serpins. Current analysis offers a new look on how proteins can change fold in the course of evolution.

Prediction of the Archaeal Exosome and Its Connections with the Proteasome and the Translation and Transcription Machineries by a Comparative-Genomic Approach

Abstract: By comparing the gene order in the completely sequenced archaeal genomes complemented by sequence profile analysis, we predict the existence and protein composition of the archaeal counterpart of the eukaryotic exosome, a complex of RNAses, RNA-binding proteins, and helicases that mediates processing and 3′->5′ degradation of a variety of RNA species. The majority of the predicted archaeal exosome subunits are encoded in what appears to be a previously undetected superoperon. In Methanobacterium thermoautotrophicum, this predicted superoperon consists of 15 genes; in the Crenarchaea, Sulfolobus solfataricus and Aeropyrum pernix, one and two of the genes from the superoperon, respectively, are relocated in the genome, whereas in other Euryarchaeota, the superoperon is split into a variable number of predicted operons and solitary genes. Methanococcus jannaschii partially retains the superoperon, but lacks the three core exosome subunits, and in Halobacterium sp., the superoperon is divided into two predicted operons, with the same three exosome subunits missing. This suggests concerted gene loss and an alteration of the structure and function of the predicted exosome in the Methanococcus and Halobacterium lineages. Additional potential components of the exosome are encoded by partially conserved predicted small operons. Along with the orthologs of eukaryotic exosome subunits, namely an RNase PH and two RNA-binding proteins, the predicted archaeal exosomal superoperon also encodes orthologs of two protein subunits of RNase P. This suggests a functional and possibly a physical interaction between RNase P and the postulated archaeal exosome, a connection that has not been reported in eukaryotes. In a pattern of apparent gene loss complementary to that seen in Methanococcus and Halobacterium, Thermoplasma acidophilum lacks the RNase P subunits. Unexpectedly, the identified exosomal superoperon, in addition to the predicted exosome components, encodes the catalytic subunits of the archaeal proteasome, two ribosomal proteins and a DNA-directed RNA polymerase subunit. These observations suggest that in archaea, a tight functional coupling exists between translation, RNA processing and degradation, (apparently mediated by the predicted exosome) and protein degradation (mediated by the proteasome), and may have implications for cross-talk between these processes in eukaryotes.

Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps

Abstract: Eukaryotic ribosome maturation depends on a set of well ordered processing steps. Here we describe the functional characterization of yeast Nog2p (Ynr053cp), a highly conserved nuclear protein. Nog2p contains a putative GTP-binding site, which is essential in vivo. Kinetic and steady-state measurements of the levels of pre-rRNAs in Nog2p-depleted cells showed a defect in 5.8S and 25S maturation and a concomitant increase in the levels of both 27SB(S) and 7S(S) precursors. We found Nog2p physically associated with large pre-60S complexes highly enriched in the 27SB and 7S rRNA precursors. These complexes contained, besides a subset of ribosomal proteins, at least two additional factors, Nog1p, another putative GTP-binding protein, and Rlp24p (Ylr009wp), which belongs to the Rpl24e family of archaeal and eukaryotic ribosomal proteins. In the absence of Nog2p, the pre-60S ribosomal complexes left the nucleolus, but were retained in the nucleoplasm. These results suggest that transient, possibly GTP-dependent association of Nog2p with the pre-ribosomes might trigger late rRNA maturation steps in ribosomal large subunit biogenesis.

The Saccharomyces cerevisiae TIF6 Gene Encoding Translation Initiation Factor 6 Is Required for 60S Ribosomal Subunit Biogenesis

Abstract: Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. In Saccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.

The p70 S6 kinase integrates nutrient and growth signals to control translational capacity.

Abstract: The p70 S6 kinase was one of the first insulin/mitogen activated protein (Ser/Thr) kinases to be described. The kinase was purified to homogeneity over a decade ago, its molecular structure defined and the enzymology of its phosphorylation of the 40 S subunit protein S6 well worked out, both in vitro and in vivo. By contrast, the cellular function of the kinase, the mechanisms of activation and the nature of the signal transduction elements upstream have been elucidated only within the last several years. This review will describe our current understanding of the biologic role of the p70 kinase as gleaned from experiments in mammalian cell culture, and through gene deletion in mouse and Drosophila, as well as the regulation of the p70 kinase and the nature of the signal transduction pathways that funnel into the control of this ubiquitous signal-responsive ribosomal protein kinase

Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA.

Abstract: S1 is the largest ribosomal protein, present in the small subunit of the bacterial ribosome. It has a pivotal role in stabilizing the mRNA on the ribosome. Thus far, S1 has eluded structural determination. We have identified the S1 protein mass in the cryo-electron microscopic map of the Escherichia coli ribosome by comparing the map with a recent x-ray crystallographic structure of the 30S subunit, which lacks S1. According to our finding, S1 is located at the junction of head, platform, and main body of the 30S subunit, thus explaining all existing biochemical and crosslinking data. Protein S1 as identified in our map has a complex, elongated shape with two holes in its central portion. The N-terminal domain, forming one of the extensions, penetrates into the head of the 30S subunit. Evidence for direct interaction of S1 with 11 nucleotides of the mRNA, immediately upstream of the Shine–Dalgarno sequence, explains the protein's role in the recognition of the 5′ region of mRNA.

Two C or not two C: recurrent disruption of Zn-ribbons, gene duplication, lineage-specific gene loss, and horizontal gene transfer in evolution of bacterial ribosomal proteins

Abstract: Background: Ribosomal proteins are encoded in all genomes of cellular life forms and are, generally, well conserved during evolution. In prokaryotes, the genes for most ribosomal proteins are clustered in several highly conserved operons, which ensures efficient co-regulation of their expression. Duplications of ribosomal-protein genes are infrequent, and given their coordinated expression and functioning, it is generally assumed that ribosomal-protein genes are unlikely to undergo horizontal transfer. However, with the accumulation of numerous complete genome sequences of prokaryotes, several paralogous pairs of ribosomal protein genes have been identified. Here we analyze all such cases and attempt to reconstruct the evolutionary history of these ribosomal proteins. Results: Complete bacterial genomes were searched for duplications of ribosomal proteins. Ribosomal proteins L36, L33, L31, S14 are each duplicated in several bacterial genomes and ribosomal proteins L11, L28, L7/L12, S1, S15, S18 are so far duplicated in only one genome each. Sequence analysis of the four ribosomal proteins, for which paralogs were detected in several genomes, two of the ribosomal proteins duplicated in one genome (L28 and S18), and the ribosomal protein L32 showed that each of them comes in two distinct versions. One form contains a predicted metal-binding Zn-ribbon that consists of four conserved cysteines (in some cases replaced by histidines), whereas, in the second form, these metal-chelating residues are completely or partially replaced. Typically, genomes containing paralogous genes for these ribosomal proteins encode both versions, designated C+ and C-, respectively. Analysis of phylogenetic trees for these seven ribosomal proteins, combined with comparison of genomic contexts for the respective genes, indicates that in most, if not all cases, their evolution involved a duplication of the ancestral C+ form early in bacterial evolution, with subsequent alternative loss of the C+ and C- forms in different lineages. Additionally, evidence was obtained for a role of horizontal gene transfer in the evolution of these ribosomal proteins, with multiple cases of gene displacement ‘in situ’, that is, without a change of the gene order in the recipient genome. Conclusions: A more complex picture of evolution of bacterial ribosomal proteins than previously suspected is emerging from these results, with major contributions of lineage-specific gene loss and horizontal gene transfer. The recurrent theme of emergence and disruption of Zn-ribbons in bacterial ribosomal proteins awaits a functional interpretation.

Characterizations of Highly Expressed Genes of Four Fast-Growing Bacteria

Abstract: Predicted highly expressed (PHX) genes are characterized for the completely sequenced genomes of the four fast-growing bacteria Escherichia coli, Haemophilus influenzae, Vibrio cholerae, and Bacillus subtilis. Our approach to ascertaining gene expression levels relates to codon usage differences among certain gene classes: the collection of all genes (average gene), the ensemble of ribosomal protein genes, major translation/transcription processing factors, and genes for polypeptides of chaperone/degradation complexes. A gene is predicted highly expressed (PHX) if its codon frequencies are close to those of the ribosomal proteins, major translation/transcription processing factor, and chaperone/degradation standards but strongly deviant from the average gene codon frequencies. PHX genes identified by their codon usage frequencies among prokaryotic genomes commonly include those for ribosomal proteins, major transcription/translation processing factors (several occurring in multiple copies), and major chaperone/degradation proteins. Also PHX genes generally include those encoding enzymes of essential energy metabolism pathways of glycolysis, pyruvate oxidation, and respiration (aerobic and anaerobic), genes of fatty acid biosynthesis, and the principal genes of amino acid and nucleotide biosyntheses. Gene classes generally not PHX include most repair protein genes, virtually all vitamin biosynthesis genes, genes of two-component sensor systems, most regulatory genes, and most genes expressed in stationary phase or during starvation. Members of the set of PHX aminoacyl-tRNA synthetase genes contrast sharply between genomes. There are also subtle differences among the PHX energy metabolism genes between E. coli and B. subtilis, particularly with respect to genes of the tricarboxylic acid cycle. The good agreement of PHX genes of E. coli and B. subtilis with high protein abundances, as assessed by two-dimensional gel determination, is verified. Relationships of PHX genes with stoichiometry, multifunctionality, and operon structures are also examined. The spatial distribution of PHX genes within each genome reveals clusters and significantly long regions without PHX genes.

Structural basis for selectivity and toxicity of ribosomal antibiotics

Abstract: Ribosomal antibiotics must discriminate between bacterial and eukaryotic ribosomes to various extents Despite major differences in bacterial and eukaryotic ribosome structure, a single nucleotide or amino acid determines the selectivity of drugs affecting protein synthesis Analysis of resistance mutations in bacteria allows the prediction of whether cytoplasmic or mitochondrial ribosomes in eukaryotic cells will be sensitive to the drug This has important implications for drug specificity and toxicity Together with recent data on the structure of ribosomal subunits these data provide the basis for development of new ribosomal antibiotics by rationale drug design

Ribosomal protein S4 is a transcription factor with properties remarkably similar to NusA, a protein involved in both non-ribosomal and ribosomal RNA antitermination.

Abstract: Escherichia coli ribosomal RNA (rRNA) operons contain antitermination motifs necessary for forming terminator-resistant transcription complexes. In preliminary work, we isolated ‘antiterminating’ transcription complexes and identified four new proteins potentially involved in rRNA transcription antitermination: ribosomal (r-) proteins S4, L3, L4 and L13. We show here that these r-proteins and Nus factors lead to an 11-fold increase in terminator read-through in in vitro transcription reactions. A significant portion of the effect was a result of r-protein S4. We show that S4 acted as a general antitermination factor, with properties very similar to NusA. It retarded termination and increased read-through at Rho-dependent terminators, even in the absence of the rRNA antiterminator motif. High concentrations of NusG showed reduced antitermination by S4. Like rrn antitermination, S4 selectively antiterminated at Rho-dependent terminators. Lastly, S4 tightly bound RNA polymerase in vivo. Our results suggest that, like NusA, S4 is a general transcription antitermination factor that associates with RNA polymerase during normal transcription and is also involved in rRNA operon antitermination. A model for key r-proteins playing a regulatory role in rRNA synthesis is presented.