N-TERMINAL PROCESSING OF RIBOSOMAL PROTEIN L27 IN STAPHYLOCOCCUS AUREUS

The bacterial ribosome is essential to cell growth yet little is known about how its proteins attain their mature structures. Recent studies indicate that certain Staphlyococcus aureus bacteriophage protein sequences contain specific sites that may be cleaved by a non-bacteriophage enzyme (Poliakov et al. 2008). The phage cleavage site was found to bear sequence similarity to the N-terminus of S. aureus ribosomal protein L27. Previous studies in E. coli (Wower et al.1998; Maguire et al. 2005) found that L27 is situated adjacent to the ribosomal peptidyl transferase site, where it likely aids in new peptide formation. The predicted S. aureus L27 protein contains an additional N-terminal sequence not observed within the N-terminus of the otherwise similar E. coli L27; this sequence appears to be cleaved, indicating yet-unobserved ribosomal protein post-translational processing and use of host processes by phage. Phylogenetic analysis shows that L27 processing has the potential to be highly conserved. Further study of this phenomenon may aid antibiotic development

The Genesis of Ribosome Structure: A Tale of Two Proteins

Living cells are dependent upon protein synthesis for virtually all cellular functions. The cellular machine responsible for protein synthesis, called the ribosome, is formed through the association of two unequally sized subunits, each composed of RNA and proteins. Proper assembly of each subunit is essential to ribosome function and therefore essential to the cellular life cycle. Previous studies focused on dissecting the assembly of the small ribosomal subunit (30S subunit) from E. coli have shown that 21 proteins sequentially assemble on the 16S rRNA at multiple nucleation sites. For the first time, we are able to monitor changes in the secondary and tertiary structure of the 16S rRNA upon the addition of single proteins during assembly by using time-dependent chemical probing. Results from these studies suggest that protein S17 induces multiple structural changes in 16S rRNA by first binding to helix 11 and then helix 7. S20 also induces changes in the rRNA by interacting with helix 9, 11, 44 and 13 in that order. These structural formations and rearrangements then prepare the binding sites for additional proteins (S12 and S16, respectively). This study demonstrates that time-dependent chemical probing is able to monitor the assembly of the 30S subunit at a level of detail never before seen. These studies also suggest that many motifs in the 16S rRNA structure are formed as a result of the proteins binding, lending evidence to the hypothesis that the function of ribosomal proteins is to shape and/or hold the RNA structure in place

Reconstructing phylogeny from RNA secondary structure via simulated evolution

DNA sequences of genes encoding functional RNA molecules (e.g., ribosomal RNAs) are commonly used in phylogenetics (i.e. to infer evolutionary history). Trees derived from ribosomal RNA (rRNA) sequences, however, are inconsistent with other molecular data in investigations of deep branches in the tree of life. Since much of te functional constraints on the gene products (i.e. RNA molecules) relate to three-dimensional structure, rather than their actual sequences, accumulated mutations in the gene sequences may obscure phylogenetic signal over very large evolutionary time-scales. Variation in structure, however, may be suitable for phylogenetic inference even under extreme sequence divergence. To evaluate qualitatively the manner in which structural evolution relates to sequence change, we simulated the evolution of RNA sequences under various constraints on structural change

The Comparative RNA Web (CRW) Site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs

BACKGROUND: Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields. RESULTS: We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems. CONCLUSIONS: This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site

Ribosoomide lagundamine bakterites

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRibosoomid on makromolekulaarsed kompleksid, mis koosnevad kahest suurest ja ühest väikesest RNAst ja paljudest erinevatest valkudest. Ribosoomides sünteesitakse kõik valgud, mida organismis leida võib, ning aktiivsete ribsoomide konsentratsioon (ja seega sünteesi kiirus) limiteerib rakkude kasvu kiirust. Ehk teisisõnu, mida kiiremini sünteesitakse uusi ribosoome, seda kiiremini kasvab ja jaguneb ka rakk. Kuna ribosomaalse RNA süntees hõlmab ca 80% raku RNA sünteesi aktiivsusest ja ribosoomi valgud moodustavad kuni veerandi raku valgumassist on selge, et mitte ainult ribosoomide funktsioon valgusünteesil vaid ka nende metabolism on rakulises majapidamises määrava tähtsusega. Tõepoolest, juba mõnda aega on teada, et aeglaselt kasvavates bakterirakkudes tegeleb enamus raku RNA lagundamise võimekusest värskelt sünteesitud ribosomaalse RNA lagundamisega. Sellegipoolest on viimase 50 aasta vältel üldiselt usutud, et kord juba valmis tehtud ja kokku pakitud ribosoomid on äärmiselt stabiilsed ning, et neid lagundatakse vaid tugeva stressi tingimustes. Samuti on meie teadmised ribosoomide lagundamise molekulaarsetest mehhanismidest bakteris üsnagi piiratud. Käesoleva doktoritöö eesmärk on kirjeldada ribosoomide lagundamist kasvavates soolekepikese (Escherichia coli) rakkudes ja heita valgust ribsoomide lagundamise mehhanismidele, molekulaarsetele radadele ning ensüümidele, mis selles protsessis osalevad. Me avastasime üllatusega, et kuigi ribosoome tõepoolest lagundatakse kasvavates bakterirakkudes, toimub see protsess vaid rakukultuuri kasvu aeglustumise perioodil, mis eelneb statsionaarse kasvufaasi saabumisele. Meil ei õnnestunud tuvastada küpsete ribosoomide lagundamist ei ühtlase kiirusega kasvavates ega ka null-kiirusega kasvavates rakkudes. Võimalik, et ribosoomide lagundamine aitab rakke neid ette valmistades eluks statsionaarses faasis, mil ei vajata suurt valgusünteesi võimekust, küll aga vabu komponente, millest elutingimuste paranedes kiiresti uusi makromolekule tootma hakata. Lisaks leidsime, et osad (kuid mitte kõik) ribosoomi RNAd inaktiveerivad mutatsioonid viivad samuti ribsoomide lagundamisele, kuid miskipärast lagundatakse siis nii mutantseid ning inaktiivseid kui metsiktüüpi ning aktiivseid ribosoome. Jällegi viitab see, et ribsoomide lagundamise eesmärk võiks olla üldise ribosoomide konsentratsiooni alandamine rakus. Kui me lisasime ribsoomide lagundamise katsesüsteemi valgusünteesi pärssivat antibiootikumi kloramfenikool, päästsime me sellega ribosoomid lagundamisest. Seda tulemust võib tõlgendada viisil, et de novo valgusüntees on vajalik ribosoomide lagundamisprogrammi käivitamiseks rakus. Testides ribosoomide lagundamise võime osas bakteritüvesid, kus puuduvad erinevad RNAd lagundavad ensüümid, leidsime kaks ensüümi, mille puudumise korral ribosoome ei lagundatud. Neist esimene, RNaas R, lõhub RNAsid alates nende tagumisest ehk 3’ otsast ning tunneb erilist lõbu kõrge sekundaarstruktuuriga RNA-de hävitamisest. RNaas R on ka eelnevalt näidatud osalevat ribosoomide lagundamisel. Teine ensüüm on seevastu suhteliselt vähetuntud endoribonukleeas nimega YbeY, mis lõikab RNAd katki keskelt, mitte ei lagunda seda otstest. See huvitav valk on arvatud osalevat ribsoomide kokkupakkimise kvaliteedikontrollil, kus ta on vajalik kõige viimases etapis, mil tuntakse ära valgusünteesil ebaõnnestuvad ribosoomid ja suunatakse need lagundamisse. Meie katsed viitavad, et seesama valk võib valla päästa ka töökorras olevate ribosoomide lagundamise, tehes ribosoomi RNAsse esimese lõike ning tekitades sellega kaitsetu 3’ otsa, mida tunneb ära RNaas R, mis omakorda suudab ribosoomi RNA täielikult lagundadaRibosomes are macromolecular complexes that consist of two large and one small RNA and of many different small proteins. The ribosome synthesizes all cellular proteins and the concentration of active ribosomes is rate limiting for cell growth. As synthesis or ribosomal RNA encompasses 80% of cellular RNA synthesis activity and the ribosomal proteins can make up half of the cellular protein mass, it is clear that ribosomal metabolism, including ribosomal degradation, makes a worthy object of study. Nevertheless, during the past half century it has been widely believed that mature ribosomes are quite stable in the cells. The major goal of this dissertation is to describe the degradation of mature ribosomes in growing E. coli cells and to shed light on the molecular mechanism of degradation. We discovered that while mature ribosomes are indeed degraded in cells growing in batch cultures, this process is limited to the slowing of growth phase, which precedes entry into the stationary phase. We were unable to detect degradation during constant-rate growth and during early stationary phase. In addition, we found that some, but not all, ribosome-inactivating mutations in 23S rRNA and 16S rRNA led to degradation of both mutant and wild-type ribosomal RNAs. Thus, unlike in yeast, the ribosome degradation in E. coli is a general process that, once initiated, does not discriminate between active and inactive ribosomes. As ribosome degradation is inhibited by the protein synthesis inhibitor chloramphenicol, we further suggest that de novo protein synthesis might be needed for triggering the degradation program. To pinpoint the enzymes responsible for degradation we tested several strains defective for different RNases. We found two RNases, RNaseR and YbeY, whose deletion saved ribosomes from degradation. RNaseR is a well studied 3’ to 5’ exonuclease whose role in degrading heavily structured RNAs, including the rRNAs, is well established. In contrast YbeY is a potential endonuclease recently implicated in a late step ribosomal quality control, which could well be the initiating endonuclease, whose cut(s) in rRNA would present substrates for RNaseR to further scavenge into mononucleotides

Structure of RNA polymerase bound to ribosomal 30S subunit

In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome. The nucleic-acid-binding cleft of RNAP samples distinct conformations, suggesting different functional states during transcription-translation coupling. The architecture of the 30S*RNAP complex provides a structural basis for co-localization of the transcriptional and translational machineries, and inform future mechanistic studies of coupled transcription and translation

More than an RNA matchmaker: Expanding the roles of Hfq into ribosome biogenesis

Ribosome biogenesis is a complex process involving multiple factors. The work described here is primarily centered in the study of ribosomal RNA, highlighting its central role in translation regulation. We have uncovered new regulators involved in rRNA processing, folding and degradation pathways. For the first time, we demonstrate that the widely conserved RNA chaperone Hfq, mostly known as the sRNA-mRNA matchmaker, acts as a ribosomal assembly factor in Escherichia coli, affecting rRNA processing, ribosome levels, translation efficiency and accuracy. This function is suggested to be independent of its activity as sRNA-regulator.(...

Atomic Structures of the 30S Subunit and Its Complexes with Ligands and Antibiotics

The two subunits that make up the ribosome have both distinct and cooperative functions. The 30S ribosomal subunit binds messenger RNA (mRNA) and is involved in the selection of cognate transfer RNA (tRNA) by monitoring codon–anticodon base-pairing during the decoding process. The 50S subunit catalyzes peptide-bond formation. Both subunits work in concert to move tRNAs and mRNAs relative to the ribosome in translocation, and both are the target of a large number of naturally occurring antibiotics. Thus, useful information about the mechanism of translation can be gleaned from structures of both individual subunits and the intact ribosome. In this paper, we describe our work on the determination of the atomic structure of the 30S ribosomal subunit and its complexes with RNA ligands, antibiotics, and initiation factor IF1. The results provide structural insights into how the ribosome recognizes cognate tRNA and discriminates against near-cognate tRNA. They also provide a structural basis for understanding the action of various antibiotics that target the 30S subunit

Functional Interactions of metalloprotein YbeY, involved in ribosomal metabolism, with the putative metal efflux protein YbeX

YbeY is a putative ribosomal endoribonuclease which has been implicated, among other things, to be involved in quality control of 70S ribosomes, in 17S pre-rRNA maturation and in ribosomal degradation. However, controversy reigns over its mode of action, substrates, co-factors, and interaction partners. Proposed interactors of YbeY include ribosomal protein S11, Era, YbeZ, and SpoT. In many bacteria ybeY is located in the ybeZYX-Int operon, where ybeZ encodes a PhoH subfamily protein with NTP hydrolase domain and ybeX encodes a putative Cobalt/Magnesium efflux protein. Depletion of YbeY and YbeX have largely overlapping phenotypes, including accumulation of 17S pre-rRNA and an approximately 1 kb 16S rRNA cleavage product, sensitivity to heat shock, and to the protein synthesis inhibitors chloramphenicol and erythromycin. Overexpression of the YbeY partially rescues the some of the phenotypes of ΔybeX. Taken together our results indicate a functional interaction between ybeY and ybeX