Analysis of secondary structure within sgm and kgmB mRNA

Sgm methyltransferase from Micromonospora zionensis and KgmB methyltransferase from Streptoalloteichus tenebrarius are resistant to aminoglycoside antibiotics as a result of their ability to specifically methylate G1405 within the bacterial 16S rRNA A-site. The (C)CGCCC motif, assumed to be a regulatory sequence responsible for the autoregulation of the sgm gene, could most likely also be responsible for the autoregulation of the kgmB gene. This sequence, found within the 5' untranslated region of both sgm and kgmB mRNAs, as indicated by in silico prediction, may be involved in the formation of a specific stem-loop structure. Sgm and KgmB are mutually down-regulated and it is likely that they share the same cis-acting elements. Structure probing experiments confirmed the existence of a stable secondary structure within the 5' UTR of the sgm mRNA, while the analysis of kgmB mRNA failed to confirm the predicted structure.

Analysis of secondary structure within sgm and kgmB mRNA

Sgm metiltransferaza iz soja Micromonospora zionensis i KgmB metiltransferaza iz soja Streptoalloteichus tenebrarius ostvaruju rezistenciju na aminoglikozidne antibiotike metilacijom nukleotida na poziciji G1405 u okviru A mesta na 16S rRNK. Smatra se da je za autoregulaciju sgm gena odgovoran (C)CCGCCC motiv. Najverovatnije je ista sekvenca odgovorna i za autoregulaciju kgmB gena. Po kompjuterskoj predikciji, ovaj motiv, lociran u 5' netranslatirajućem regionu iRNK molekula oba gena, bi mogao učestvovati u formiranju sekundarne strukture tipa ukosnice. Kako Sgm i KgmB metiltransferaze jedna drugu autoregulišu, moguće je da prepoznaju iste cis elemente u iRNK molekulima. Eksperimenti ispitivanja strukture su, s jedne strane potvrdili prisustvo stabilne sekundarne strukture u okviru 5' netranslatirajućeg regiona iRNK molekula sgm gena, a sa druge, nisu dokazali postojanje modelovane sekundarne strukture u iRNK molekulu kgmB gena.Sgm methyltransferase from Micromonospora zionensis and KgmB methyltransferase from Streptoalloteichus tenebrarius are resistant to aminoglycoside antibiotics as a result of their ability to specifically methylate G1405 within the bacterial 16S rRNA A-site. The (C)CGCCC motif, assumed to be a regulatory sequence responsible for the autoregulation of the sgm gene, could most likely also be responsible for the autoregulation of the kgmB gene. This sequence, found within the 5' untranslated region of both sgm and kgmB mRNAs, as indicated by in silico prediction, may be involved in the formation of a specific stem-loop structure. Sgm and KgmB are mutually down-regulated and it is likely that they share the same cis-acting elements. Structure probing experiments confirmed the existence of a stable secondary structure within the 5' UTR of the sgm mRNA, while the analysis of kgmB mRNA failed to confirm the predicted structure

Translational Repression of Bacteriophage T4 DNA Polymerase Biosynthesis

The research described in this dissertation elucidated the mechanism by which bacteriophage T4 DNA polymerase regulates its own biosynthesis. Utilizing both in vivo and in vitro studies, I have shown that autogenous repression occurs at the level of translation. While T4 mutants defective in the structural gene for DNA polymerase (gene 43) overproduce the protein product (gp43) in vivo, they do not overproduce the corresponding mRNA. In vitro, purified DNA polymerase specifically inhibited the translation of its own transcripts. Further, it was demonstrated that gp43 binds its own mRNA at a site overlapping the ribosome initiation domain. Thus, T4 DNA polymerase is a specific translational repressor that presumably inhibits initiation of translation. The mRNA binding site (translational operator) for DNA polymerase includes 38-40 nucleotides upstream of the initiator AUG. The 5\u27 half of this translational operator contains a putative five base-pair stem and 8-base loop, whose existence is inferred from RNase digestion experiments and computer-assisted analysis of RNA folding. To ascertain the important RNA sequence and structural determinants for DNA polymerase binding, I carried out a mutational analysis of the translational operator via the in vitro construction of several operator variants. Operator mutants were subsequently assayed for the effect of each mutation on: 1) gp43/mRNA binding, in vitro 2) the in vivo levels of gp43 biosynthesis from plasmid encoded constructs and 3) in vivo level of gp43 synthesis in phage infections (carried out after introducing mutant operators into the phage genome by virus-plasmid recombination). Mutations that either disrupted the stem or altered particular loop residues, led to diminished binding of purified T4 DNA polymerase in vitro and to derepression of protein synthesis in vivo. Compensatory mutations that restored the stern pairing, with a sequence other than wild-type, restored in vitro binding but still exhibited a mutant phenotype in vivo. Results from loop substitutions suggest that the spatial arrangement of specific loop residues is a major criterion for specific binding of DNA polymerase to its mRNA operator. These studies demonstrate the effectiveness of genetic approaches in dissecting the rules that govern RNA-protein interactions

The KsgA methyltransferase: Characterization of a universally conserved protein involved in robosome biogenesis

The KsgA enzymes comprise an ancient family of methyltransferases that are intimately involved in ribosome biogenesis. Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout evolution, and therefore were present in the last common ancestor. The first member of the family to be described, KsgA from Escherichia coli, was initially shown to be the determining factor for resistance/sensitivity to the antibiotic kasugamycin and was subsequently found to dimethylate two adenosines in 16S rRNA during maturation of the 30S subunit. Since then, numerous other members of the family have been characterized in eubacteria, eukaryotes, archaea and in eukaryotic organelles. The eukaryotic ortholog, Dim1, is essential for proper processing of the pre-rRNA, in addition to and separate from its methyltransferase function. The KsgA/Dim1 family bears sequence and structural similarity to a larger group of S-adenosyl-L-methionine dependent methyltransferases, which includes both DNA and RNA methyltransferases. In this document we report that KsgA orthologs from archaea and eukaryotes are able to complement for KsgA function in bacteria, both in vivo and in vitro. This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life. We have characterized KsgA structurally, and discuss aspects of KsgA\u27s activity in light of the structural data. We also propose a model for KsgA binding to the 30S subunit, based on solution probing data. This model sheds light on KsgA\u27s unusual regulation and on the dual function of the Dim1 enzymes

Ribosoomiga seonduvad antibiootikumid ja antibiootikumiresistentsuse mehhanismid

Väitekirja elektrooniline versioon ei sisalda publikatsiooneValgusüntees võib olla reguleeritud polüpeptiidahela poolt, mida ribosoom parajasti sünteesib. Teatud peptiidide transleerimine põhjustab ribosoomi seiskumist, mis omakorda takistab allavoolu paiknevate geenide ekspresseerumist. Teatud juhtudel vajatakse seisaku toimumiseks väikese ligandmolekuli (antibiootikum, aminohape vms) juuresolekut. Tuntakse mitmeid ribosoomi seisakut põhjustavaid peptiide, kuid nende võrdlemisel pole leitud ühtset konsensusjärjestust. Samas võib oletada, et reeglite tuvastamine on võimalik, kui analüüsida suuremat hulka peptiide. Seetõttu töötasime välja selektsioonimeetodi ribosoomi peatavate peptiide leidmiseks. Oma meetodit kasutades identifitseerisime järjestused, mis peatasid translatsiooni erütromütsiini, troleandomütsiini, klooramfenikooli, meta-toluaadi või homoseriinlaktooni juuresolekul. Need järjestused olid aktiivsed peptiidi tasemel. Me ei tuvastanud peptiidide aminohappeliste järjestuste võrdlemisel universaalset konsensusjärjestust. Siiski märkasime teatud seaduspärasusi. Näiteks selgus, et erütromütsiini juuresolekul toimuv ribosoomi seiskumine vajab eelistatult hüdrofoobset kasvavat peptiidi. On võimalik, et ribosoomi peatumist põhjustavate järjestusmotiivide leidmiseks tuleb analüüsida suuremat peptiidide valimit. Samuti avastasime, et seisakut põhjustavate peptiidide funktsionaalsus võib olla mõjutatud vastava mRNA omaduste poolt. Meie meetod võib olla rakendatav biotehnoloogias. Teoreetiliselt võiks ribosoomi peatumist põhjustavaid peptiide kasutada selleks, et luua uudseid geeniekspressiooni süsteeme, milles regulatsioon toimub translatsiooni tasemel. Väitekirja teine publikatsioon kirjeldab antibiootikumiresistentsuse mehhanismi, mis toimub ribsoomi kaitsevalkude vahendusel ja tagab resistentsuse tetratsükliinile. Töö aluseks oli ribosoomi ja kaitsevalgu Tet(O) kompleksi atomaarne mudel. Struktuuriuuringud näitasid, et Tet(O) interakteerub nii väikese kui suure ribosoomi alaühikuga. Me tegime asendus- ja deletsioonimutatsioone Tet(O) domään IV lingudesse 465, 438 ja 507, mis mudeli kohaselt interakteeruvad otseselt tetratsükliini seondumiskohaga või asuvad selle lähedal. Mutantsete ja metsik-tüüpi Tet(O) variantide analüüs näitas, et kõik kolm lingu on Tet(O) tööks hädavajalikud. Asendusmutatsioonid lingudes vähendasid Tet(O) aktiivsust ja deletsioonid kaotasid aktiivsuse täielikult. Struktuursete uuringute ja mutatsioonanalüüsi tulemusi kõrvutades pakkusime välja mudeli Tet(O) vahendusel toimuva tetratsükliiniresistentsuse mehhanismi selgitamiseks. Meie arvates muudab Tet(O) ling 465 tetratsükliini seondumiskoha juures 16S rRNA struktuuri. See nõrgendab interaktsiooni tetratsükliini molekuli ja 16S rRNA vahel ning võimaldab lingul 507 ravim ribosoomilt eemale tõugata. Me oletasime, et ling 438 koos 16S rRNA nukleotiididega moodustab kanali, mille kaudu tetratsükliin ribosoomilt lahkub. Väitekirja kolmas publikatsioon puudutab probleeme, mis on seotud uute turule ilmuvate antibiootikumidega. Nende hulgas leidub ravimeid, mille toimemehhanism pole veel täiesti selge. Üheks selliseks näiteks on nitrovinüülfuraanide hulka kuuluv G1 ehk Furvina®. G1 laguneb vees ja tioolrühmi sisaldavate ühendite (näiteks tsüsteiini) olemasolu keskkonnas kiirendab seda protsessi. Tuumamagnetresonantsi ja kõrgefektiivse vedelikukromatograafia meetodeid kasutades selgitasime välja G1 lagunemise reaktsiooniskeemi ja selle käigus tekkivad ühendid. Leidsime, et G1 antibakteriaalne toime põhineb valkudes sisalduvate tioolrühmade modifitseerimisel. On teada, et vähemalt mõned G1 lagunemise käigus tekkivatest ühenditest säilitavad antimikroobse aktiivsuse. Selleks, et teha vahet G1 ja selle laguproduktide põhjustatud antimikroobsetel efektidel, inkubeerisime uuritavat ainet enne antimikroobse aktiivsuse mõõtmist erinevates vedelsöötmetes. Selgus, et tsüsteiinivabas söötmes on G1 antimikroobne võimekus suurem kui tioolrühmi sisaldavas söötmes. Samas jäid antimikroobsed omadused alles isegi pärast kahe tunni pikkust testile eelnenud inkubatsiooniaega. Me järeldame, et G1 aktiivsus on aine kohese reaktiivsuse ja laguproduktide antimikroobsete aktiivsuste summa. Tulemused näitavad, et G1 võib sobida jagunevate bakterirakkude kasvu takistamiseks, kuid ei ole efektiivne vahend mittejagunevate bakterite vastu.Protein synthesis can be regulated by the nascent polypeptide chain currently translated by the ribosome. Translation of certain peptides causes ribosome stalling and, thus, inhibits expression of downstream genes. In some cases, stalling requires the presence of a small ligand molecule (an antibiotic, an amino acid, etc). Several peptides are known that can induce ribosome stalling, but their comparison has not revealed a common consensus motif. However, it can be assumed that the identification of rules is possible by analysing a larger number of peptides. Therefore, we developed a selection method for identifying peptides capable of stalling the ribosome. By using our method, we identified sequences that stalled translation in response to erythromycin, troleandomycin, chloramphenicol, meta-toluate or homoserine lactone. These sequences were active on the peptide level. Comparison of amino acid sequences of peptides did not reveal any universal consensus motif. However, we noticed certain tendencies. For instance, it was found that ribosome stalling in the presence of erythromycin preferably requires a hydrophobic nascent peptide. We also discovered that the functionality of stalling peptides can be affected by properties of corresponding mRNA. Our method may be applicable in biotechnology. In theory, stalling peptides could be used for designing novel gene expression systems that are regulated at the level of translation. The second publication of dissertation describes the mechanism of antibiotic resistance that is mediated by ribosome protection proteins and confers resistance to tetracycline. The work was based on the atomic model of the complex of ribosome and protection protein Tet(O). Structural studies showed that Tet(O) interacts with both small and large ribosomal subunit. We introduced substitution and deletion mutations into Tet(O) domain IV loops 465, 438 and 507 which, according to the model, interact directly with the tetracycline binding site or are located in the vicinity. Analysis of mutant and wild-type Tet(O) variants showed that all three loops are essential for Tet(O) function. Substitution mutations reduced the activity of Tet(O) and deletions abolished the activity completely. Considering the results of structural studies and mutation analysis, the model of Tet(O)-mediated tetracycline resistance mechanism was proposed. We suggest that the loop 465 of Tet(O) distorts the backbone shape of the 16S rRNA at the tetracycline-binding site. This weakens the interaction between the tetracycline molecule and 16S rRNA and enables the 507-loop to dislodge the drug from the ribosome. We hypothesized that the 438-loop along with nucleotides of 16S rRNA forms a corridor allowing tetracycline to leave the ribosome. The third part of thesis concerns problems that are related to new antibiotics emerging on the market. Among them, there are drugs whose mechanism of action is not yet completely understood. One such example is a nitrovinylfuran derivative G1, also known as Furvina®. G1 decomposes in aqueous media and the presence of thiol-containing compounds (e.g. cysteine) accelerates this process. Nuclear magnetic resonance and high performance liquid chromatography were used to clarify the reaction scheme of G1 decomposition along with the decomposition products. We found that the antibacterial effect of G1 is based on the modification of thiol groups present in proteins. It is known that at least some of G1 decomposition products retain antimicrobial activity. In order to distinguish between the effects of G1 and its conversion products, we preincubated the test substance in different liquid growth media prior to measurement of microbial activity. We found that the antimicrobial capability of G1 is greater in cysteine-free medium when compared to media containing thiol groups. However, the antimicrobial properties were not lost even after two hours of preincubation. We conclude that the activity of G1 is a sum of its immediate reactivity and effects of its breakdown products. Our data suggest that G1 can be suitable for preventing the growth of proliferating bacterial cells, but it is not an effective tool against non-growing bacteria

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

Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria

Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a > 10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs

Mutaciones en genes modificadores de ARN ribosómico y la resistencia a aminoglucósidos: el caso del gen rsmG

Introduction: Aminoglycosides like streptomycin are well-known for binding at specific regions of ribosome RNA and then acting as translation inhibitors. Nowadays, several pathogens have been detected to acquire an undefined strategy involving mutation at non structural ribosome genes like those acting as RNA methylases. rsmG is one of those genes which encodes an AdoMet-dependent methyltransferase responsible for the synthesis of m7G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m7G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning.Objectives: After taking into account genetic information indicating that some clinical isolates of human pathogens show streptomycin resistance associated with mutations at rsmG, we decided to explore new hot spots for mutation capable of impairing the RsmG in vivo function and of promoting low-level streptomycin resistance.Materials and methods: To gain insights into the molecular and genetic mechanism of acquiring this aminoglycoside resistance phenotype and the emergence of high-level streptomycin resistance in rsmG mutants, we mutated Escherichia coli rsmG and also performed a genotyping study on rpsL from several isolates showing the ability to grow at higher streptomycin concentrations than parental strains.Results: We found that the mutations at rpsL were preferentially present in these mutants, and we observed a clear synergy between rsmG and rpsL genes to induce streptomycin resistance.Conclusion: We contribute to understand a common mechanism that is probably transferable to other ribosome RNA methylase genes responsible for modifications at central sites for ribosome function.Introducción. Los aminoglucósidos son moléculas antibióticas capaces de inhibir la síntesis de proteínas bacterianas tras su unión al ribosoma procariota. La resistencia a aminoglucósidos está clásicamente asociada a mutaciones en genes estructurales del ribosoma bacteriano; sin embargo, varios estudios recientes han demostrado, de forma recurrente, la presencia de un nuevo mecanismo dependiente de mutación que no involucra genes estructurales. El gen rsmG es uno de ellos y se caracteriza por codificar una metiltransferasa que sintetiza el nucleósido m7G527 localizado en el loop 530 del ribosoma bacteriano, este último caracterizado como sitio preferencial al cual se une la estreptomicina.Objetivo. Partiendo de las recientes asociaciones clínicas entre las mutaciones en el gen rsmG y la resistencia a estreptomicina, este estudio se propuso la caracterización de nuevos puntos calientes de mutación en este gen que puedan causar resistencia a estreptomicina usando Escherichia coli como modelo de estudio.Materiales y métodos. Se indagó sobre el mecanismo genético y molecular por el cual se adquiere la resistencia a estreptomicina y su transición a la resistencia a altas dosis mediante mutagénesis dirigida del gen rsmG y genotipificación del gen rpsL.Resultados. Se encontró que la mutación N39A en rsmG inactiva la proteína y se reportó un nuevo conjunto de mutaciones en rpsL que confieren resistencia a altas dosis de estreptomicina.Conclusiones. Aunque los mecanismos genéticos subyacentes permanecen sin esclarecer, se concluyó que dichos patrones secuenciales de mutación podrían tener lugar en otros genes modificadores del ARN bacteriano debido a la conservación evolutiva y al papel crítico que juegan tales modificaciones en la síntesis de proteínas