Binding characteristics and localization of Arabidopsis thaliana ribosomal protein S15a isoforms

Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly

Cell-Free Protein Synthesis

The Nobel Prize in Medicine 1968 for interpretation of the genetic code and its function in protein synthesis and in Chemistry 2009 for studies of the structure and function of the ribosome highlighted the ground-breaking experiment performed on May 15, 1961 by Nirenberg and Matthaei and their principal breakthrough on the creation of "cell-free protein synthesis (CFPS) system". Since then the continuous technical advances have revitalized CFPS system as a simple and powerful technology platform for industrial and high-throughput protein production. CFPS yields exceed grams protein per liter reaction volume and offer several advantages including the ability to easily manipulate the reaction components and conditions to favor protein synthesis, decreased sensitivity to product toxicity, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and suitability for miniaturization and high-throughput applications. With these advantages, there is continuous increasing interest in CFPS system among biotechnologists, molecular biologists and medical or pharmacologists

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

Selection and characterization of RNA aptamers that detect a quaternary structure for ribosomal protein S7

Here we report on the selection and characterization of RNA aptamers that recognize E. coli ribosomal protein S7. Ribosomal protein S7 plays two important roles in ribosome biogenesis: (1) as an assembly initiator, S7 nucleates the folding of the 3\u27 major domain of 16S rRNA, and (2) it binds to the str operon and represses the translation of S12, S7, and EF-G. The primary and secondary structures of the S7 binding sites of rRNA and mRNA share limited sequence and structural homology and the required elements for high affinity binding have not been entirely elucidated. We have selected RNA aptamers that share very little primary sequence homology to either the S7 binding site of 16S rRNA or to the intercistronic region of str mRNA. Many of the aptamers are expected to fold into three-helix junctions, a structure particularly reminiscent of the mRNA. Interestingly, the aptamers exhibit cooperative binding with Hill coefficients of ~3 indicating that they are detecting a quaternary structure of S7. We have found that the S7 aptamers use the same amino acids and structural elements to bind S7 as the rRNA and mRNA indicating that the same binding site is used for all three RNAs. With gel filtration, we were only able to isolate the aptamer/S7 complex at a 1:1 stoichiometry, indicating that the proposed quaternary structure of S7 is weak. However, deletion of the β-ribbon nearly eliminates cooperative aptamer binding suggesting that this structural element may be involved in protein-protein interaction. Furthermore, pre-treatment of native S7 with the N-terminal extension also results in a significant reduction in cooperative aptamer binding. The results presented here suggest that S7 itself may undergo conformational rearrangement subsequent to 16S rRNA binding, and may help explain the strong temperature-dependent rearrangements at the binding site of S7 within the 16S rRNA. Furthermore, we propose that the weak, multimeric interaction of S7 may have a role in the retroregulation of S12. S7 may bind to the mRNA in a pre-multimerized form or multimerize subsequent to binding, resulting in ribosome stalling due to the multimeric obstacle. If the S7/S7 interaction is weak however, then it may be easily disrupted by repeated ribosome bombardment, causing eventual decay of the multimer and relieving some of the translational repression. Translational repression of the genes encoding S7 and EF-G would remain constant over time however, because the monomeric S7 bound more tightly to the intercistronic region would continue to prevent translational coupling with the upstream gene encoding S12

Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions

AbstractIn an attempt to understand how Escherichia coli ribosomes recognize the initiator codon on mRNAs lacking the Shine-Dalgamo (SD) sequence, we have studied 30S initiation complex formation in extension inhibition (toeprinting) experiments using (-SD)mRNAs which are known to be reliably translated in E. coli: the plant viral messenger A1MV RNA 4 and two chimaeric mRNAs coding for β-glucuronidase (GUS) and bearing the 5'-untranslated sequence of TMV RNA Ω or the Ω-derived sequence (CAA)n as 5'-leaders. Ribosomal protein Sl and IF3 have been found to be indispensable for translational initiation. Protein S1 appears to be a key recognition element. S1 binds to sequences within the leaders of (-SD)mRNAs thus providing their affinity to E. coli ribosomes

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.(...

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

Translational regulation in mycobacteria and its implications for pathogenicity.

Protein synthesis is a fundamental requirement of all cells for survival and replication. To date, vast numbers of genetic and biochemical studies have been performed to address the mechanisms of translation and its regulation in Escherichia coli, but only a limited number of studies have investigated these processes in other bacteria, particularly in slow growing bacteria like Mycobacterium tuberculosis, the causative agent of human tuberculosis. In this Review, we highlight important differences in the translational machinery of M. tuberculosis compared with E. coli, specifically the presence of two additional proteins and subunit stabilizing elements such as the B9 bridge. We also consider the role of leaderless translation in the ability of M. tuberculosis to establish latent infection and look at the experimental evidence that translational regulatory mechanisms operate in mycobacteria during stress adaptation, particularly focussing on differences in toxin-antitoxin systems between E. coli and M. tuberculosis and on the role of tuneable translational fidelity in conferring phenotypic antibiotic resistance. Finally, we consider the implications of these differences in the context of the biological adaptation of M. tuberculosis and discuss how these regulatory mechanisms could aid in the development of novel therapeutics for tuberculosis