Bakteri ribosoomi modifitseeritud nukleosiidid

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Ribosoomid on imepisikesed masinad, mis valmistavad geenides paikneva informatsiooni alusel valke. Ribosoomid on kõigis elusrakkudes üldehituselt sarnased kuigi detailides on ka palju erinevusi. Ribosoomid on keerulise ehitusega, koosnevad ribonukleiinhappest (rRNA) ja valkudest, kusjuures rRNA moodustab suurema osa ribosoomide massist. Päristuumsete organismide, sealhulgas ka inimeste, ribosoomid on suuremad ja keerulisema ehitusega kui bakterite ribosoomid, ent ribosoomide põhielemendid ja töömehanism on samad kõikides elusolendites. Kuna bakterite uurimine on tehniliselt ja eetiliselt lihtsam, on suurem osa informatsiooni ribosoomide ehituse ja töömehanismi aga ka ribosoomide enda valmistamise kohta saadud just bakteritest. rRNA koosneb pikast ribonukleosiidide (adenosiinist, guanosiinist, uratsiilist ning tsütosiinist) ahelatest. Ribosoomide sünteesimise käigus muudetakse mõningate ribonukleosiidide omadusi spetsiaalsete valkude, modifikatsiooniensüümide, poolt. Muudetud omadustega nukleosiide kutsutakse modifitseeritud nukleosiidideks. Modifitseeritud nukleosiidid esinevad kõikides ribosoomides ning paiknevad ribosoomi talitluse seisukohalt olulistes piirkondades, ent nende tähtsus ribosoomide töö seisukohalt on teadmata. Meie tuvastasime modifikatsiooniensüümi, RlmH, mis on eriline selle poolest, et modifitseerib soolekepikese (Escherichia coli) ribosoomi suurema alamühiku olulises piirkonnas paiknevat pseudouridiini, mis on omakorda modifitseeritud nukleosiid. Lisaks on RlmH huvitav selle poolest, et modifitseerib juba praktiliselt valminud ning võimalik et juba valku tootvat ribosoomi. RlmH on teadaolevalt ainuke valk, mis vajab ribosoomi ühe alamühiku rRNA modifitseerimiseks mõlemat alamühikut. Meie oleme iseloomustanud RlmH töö jaoks vajalikke tingimusi ja oleme selgitanud mehanisme, mis tagavad RlmH valgu erilisuse.Ribosomes are tiny machines that make proteins using the information stored in genes. The structure and mechanism of ribosomes is similar in all cells, but bacterial and eukaryotic (including human) ribosomes differ in details, eukaryotic ones are bigger, for instance. Ribosomes have a complicated structure; they are made of a large subunit and a small subunit both of which are made of ribonucleic acids (rRNA) and proteins. rRNA gives most of the mass to the ribosomes. Since it is both technically and ethically easier to study bacteria, most of the information about the structure and mechanisms of ribosomes as well as making the ribosomes themselves comes from them. The rRNA molecules are long chains put together of ribonucleosides (adenosine, guanosine, cytidine, and uridine). However, during making of the ribosomes but after the rRNA chain is already put together, some of the nucleosides in the chain are altered by specific proteins called modification enzymes. The altered nucleosides are called modified nucleosides and they are present in important parts of all ribosomes. However, the role of the modified nucleosides is unknown for the most part. We found that that the bacterial (Escherichia coli) modification enzyme RlmH is special because it modifies pseudouridine, which already is a modified nucleoside and is located in a very important part of the large subunit. In addition, RlmH is special because it is the only modification enzyme that requires both subunits of the ribosome to modify rRNA. In fact, RlmH modifies an almost finished ribosome that is possibly already making proteins. We have studied the conditions that RlmH requires to modify the rRNA and the mechanism behind its uniqueness

Ribosome Protection Proteins—“New” Players in the Global Arms Race with Antibiotic-Resistant Pathogens

Bacteria have evolved an array of mechanisms enabling them to resist the inhibitory effect of antibiotics, a significant proportion of which target the ribosome. Indeed, resistance mechanisms have been identified for nearly every antibiotic that is currently used in clinical practice. With the ever-increasing list of multi-drug-resistant pathogens and very few novel antibiotics in the pharmaceutical pipeline, treatable infections are likely to become life-threatening once again. Most of the prevalent resistance mechanisms are well understood and their clinical significance is recognized. In contrast, ribosome protection protein-mediated resistance has flown under the radar for a long time and has been considered a minor factor in the clinical setting. Not until the recent discovery of the ATP-binding cassette family F protein-mediated resistance in an extensive list of human pathogens has the significance of ribosome protection proteins been truly appreciated. Understanding the underlying resistance mechanism has the potential to guide the development of novel therapeutic approaches to evade or overcome the resistance. In this review, we discuss the latest developments regarding ribosome protection proteins focusing on the current antimicrobial arsenal and pharmaceutical pipeline as well as potential implications for the future of fighting bacterial infections in the time of “superbugs.

Ribosome protection proteins - “new” players in the global arms race with antibiotic-resistant pathogens

Bacteria have evolved an array of mechanisms enabling them to resist the inhibitory effect of antibiotics, a significant proportion of which target the ribosome. Indeed, resistance mechanisms have been identified for nearly every antibiotic that is currently used in clinical practice. With the ever-increasing list of multi-drug-resistant pathogens and very few novel antibiotics in the pharmaceutical pipeline, treatable infections are likely to become life-threatening once again. Most of the prevalent resistance mechanisms are well understood and their clinical significance is recognized. In contrast, ribosome protection protein-mediated resistance has flown under the radar for a long time and has been considered a minor factor in the clinical setting. Not until the recent discovery of the ATP-binding cassette family F protein-mediated resistance in an extensive list of human pathogens has the significance of ribosome protection proteins been truly appreciated. Understanding the underlying resistance mechanism has the potential to guide the development of novel therapeutic approaches to evade or overcome the resistance. In this review, we discuss the latest developments regarding ribosome protection proteins focusing on the current antimicrobial arsenal and pharmaceutical pipeline as well as potential implications for the future of fighting bacterial infections in the time of “superbugs.”Ministry of Education (MOE)Published versionThis research was funded by Ministry of Education of Singapore Tier I Grant RG108/20 (to Y.-G.G.)

Similarity and diversity of translational GTPase factors EF-G, EF4, and BipA: From structure to function

EF-G, EF4, and BipA are members of the translation factor family of GTPases with a common ribosome binding mode and GTPase activation mechanism. However, topological variations of shared as well as unique domains ensure different roles played by these proteins during translation. Recent X-ray crystallography and cryo-electron microscopy studies have revealed the structural basis for the involvement of EF-G domain IV in securing the movement of tRNAs and mRNA during translocation as well as revealing how the unique C-terminal domains of EF4 and BipA interact with the ribosome and tRNAs contributing to the regulation of translation under certain conditions. EF-G, EF-4, and BipA are intriguing examples of structural variations on a common theme that results in diverse behavior and function. Structural studies of translational GTPase factors have been greatly facilitated by the use of antibiotics, which have revealed their mechanism of action.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio

Ribosome protection by ABC‐F proteins — molecular mechanism and potential drug design

Members of the ATP‐binding cassette F (ABC‐F) proteins confer resistance to several classes of clinically important antibiotics through ribosome protection. Recent structures of two ABC‐F proteins, Pseudomonas aeruginosa MsrE and Bacillus subtilis VmlR bound to ribosome have shed light onto the ribosome protection mechanism whereby drug resistance is mediated by the antibiotic resistance domain (ARD) connecting the two ATP binding domains. ARD of the E site bound MsrE and VmlR extends toward the drug binding region within the peptidyl transferase center (PTC) and leads to conformational changes in the P site tRNA acceptor stem, the PTC, and the drug binding site causing the release of corresponding drugs. The structural similarities and differences of the MsrE and VmlR structures likely highlight an universal ribosome protection mechanism employed by antibiotic resistance (ARE) ABC‐F proteins. The variable ARD domains enable this family of proteins to adapt the protection mechanism for several classes of ribosome‐targeting drugs. ARE ABC‐F genes have been found in numerous pathogen genomes and multi‐drug resistance conferring plasmids. Collectively they mediate resistance to a broader range of antimicrobial agents than any other group of resistance proteins and play a major role in clinically significant drug resistance in pathogenic bacteria. Here, we review the recent structural and biochemical findings on these emerging resistance proteins, offering an update of the molecular basis and implications for overcoming ABC‐F conferred drug resistance

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem–loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent KM for substrate 70S ribosomes is 0.51 ± 0.06 μM, and for cofactor S-adenosyl-L-methionine 27 ± 3 μM; the kcat values are 4.95 ± 1.10 min−1 and 6.4 ± 1.3 min−1, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The KM value for substrate 50S subunits is 0.98 ± 0.18 μM and the kcat value is 1.97 ± 0.46 min−1. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD- and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits

Identification of pseudouridine methyltransferase in Escherichia coli

In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem–loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m3Ψ) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Ψ1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m3Ψ1915 is the only methylated pseudouridine in bacteria described to date