Tuning ribosomal elongation cycle by mutagenesis of 23S rRNA

http://www.ester.ee/record=b1053388~S58*es

Stability of the ‘L12 stalk’ in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria

The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called ‘L12 stalk’ on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the ‘L12 stalk’. The ‘L12 stalk’ plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L12(4) complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent

The roles of RNA helicases and other ribosome biogenesis factors during small subunit maturation

RNA helicases are a highly conserved family of proteins that act as RNA-dependent NTPases. These proteins contain a conserved helicase core consisting of two RecA-like domains that are responsible for unwinding or annealing RNA duplexes and remodelling RNP complexes in an NTP-dependent manner. As most RNA helicases perform their unwinding activity in a sequence independent manner, protein-protein interactions with cofactors can modulate their activity or provide substrate specificity. In line with their molecular functions, these proteins are central players in important cellular processes involving RNA, including pre-mRNA splicing, translation and ribosome biogenesis. The production of mature eukaryotic ribosomes is a highly dynamic and energy-consuming process that involves four rRNAs, ~80 ribosomal proteins and more than 200 trans-acting factors. In the yeast S. cerevisiae, 21 RNA helicases are involved in the assembly steps of the small and large subunits (SSU and LSU respectively), where general roles have been attributed to RNA helicases in remodelling rRNAs and modulating the dynamics of small nucleolar (sno)RNPs on pre-ribosomes. In recent years, the identification of binding sites of different RNA helicases on the rRNA as well as structural analyses of preribosomal particles has facilitated a deeper understanding of how these proteins act in ribosome biogenesis. However, for other RNA helicases, the lack of information regarding their rRNA binding sites and their molecular targets has prevented further characterisation of their functions in ribosome biogenesis. This study focused on the uncharacterised DEAD-box helicase Fal1 and the MIF4G domain-containing protein Sgd1, which are both required for SSU maturation. Analyses of pre-rRNA processing upon protein depletion demonstrated that Fal1 and Sgd1 are both required for early pre-rRNA cleavages at sites A0, A1 and A2, and complementation experiments showed that the ATPase activity of the helicase is required for this function. Fal1 and Sgd1 were shown to associate in vivo, and in vitro analyses determined that the MIF4G domain of Sgd1 mediates the interaction with Fal1. Excitingly, the data suggest that the MIF4G domain of Sgd1 can stimulate the ATPase activity of Fal1 in vitro, suggesting a role of Sgd1 as an MIF4G domain-containing cofactor of Fal1. The UV crosslinking and analysis of cDNA (CRAC) approach allowed the identification of a Sgd1 binding site within the 18S rRNA sequence, which is in line with a suggested role in the early stages of pre-SSU assembly. Interestingly, expression of different Sgd1 truncations for in vivo crosslinking experiments highlighted the C-terminal region of Sgd1 as responsible for the association with RNA. Anisotropy experiments demonstrated that the C-terminal region of Sgd1 can bind RNA in vitro in a non-sequence specific manner, suggesting that Sgd1 can simultaneously bind Fal1 through the MIF4G domain and the rRNA through the C-terminal region. Altogether, these findings expand our understanding of the role of Fal1 and Sgd1 in ribosome biogenesis, and suggest a common function of these proteins in the early stages of ribosome assembly, likely as an RNA helicasecofactor complex

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.(...

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

Elucidating Ribosomes-Genetic Studies of the ATPase Uup and the Ribosomal Protein L1.

The ribosome is comprised of two subunits, formed from three ribosomal RNAs and many ribosomal proteins. The two subunits come together around a messenger RNA during initiation, and catalyze the addition of sequential amino acids to a growing polypeptide chain as directed by the messenger RNA during elongation. While much of the ribosome is a relatively rigid molecular machine, there are two flexible protrusions comprised of rRNA and ribosomal proteins. One of these protrusions is the L1 arm where deacylated tRNAs leave the ribosome. The contributions of the L1 stalk and associating factors are unclear. In this work, I report that loss of the protein L1 results in changes in translation accuracy and perturbation of the ribosome profile. I also find that proteins involved in RpoS regulation/regulon, proteins affecting the structure of the ribosome in the vicinity of the active site, and proteins affecting DNA replication are able to suppress, when at high copy, the previously-­‐reported slow growth the of ΔrplA (encoding L1) mutant. Recent studies have revealed that translation factors act on the L1 arm side of the ribosome. I have characterized Uup, an E. coli ABCF protein that shares many of the residues involved in binding to tRNAfMet and ribosomal protein L1 in the closely related E. coli ABCF protein EttA. Exogenous Uup suppresses many phenotypes caused by deletion of tthe elongation family GTPase BipA, including a growth-­dependent ribosome profile defect. Both translation of reporter plasmids and translating ribosomes are reduced in Δuup. While Δuup and ΔettA show different phenotypes in multiple assays, each can suppress defects resulting from loss of the other. I propose that Uup, as shown for EttA, can promote formation of the first peptide bond in the elongation phase of translation. This adds to the short but rapidly growing list of translation factors regulating translation at the E­‐site.PHDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116786/1/katharyn_1.pd

RIBOSOME IN THE BALANCE: STRUCTURAL EQUILIBRIUM ENSURES TRANSLATIONAL FIDELITY AND PROPER GENE EXPRESSION

At equilibrium, empty ribosomes freely transit between the rotated and un-rotated states. In translation elongation, the binding of two translation elongation factors to the same general region of the ribosome stabilizes them in one of the two extremes of intersubunit rotation; rotated or unrotated. These stabilized states are resolved by expenditure energy in the form of GTP hydrolysis. Here, mutants of the early assembling integral ribosomal protein uL2 (universal L2) are used to test the generality of this hypothesis. A prior study employing mutants of a late assembling peripheral ribosomal protein suggested that ribosome rotational status determines its affinity for elongation factors, and hence translational fidelity and gene expression. rRNA structure probing analyses reveal that mutations in the uL2 B7b bridge region shift the equilibrium towards the rotated state, propagating rRNA structural changes to all of the functional centers of ribosome. Shift in structural equilibrium affects the biochemical properties of ribosomes: rotated ribosomes favor binding of the eEF2 translocase and disfavor that of the elongation ternary complex. This manifests as specific translational fidelity defects, impacting the expression of genes involved in telomere maintenance. A model is presented here describing how cyclic intersubunit rotation ensures the unidirectionality of translational elongation, and how perturbation of rotational equilibrium affects specific aspects of translational fidelity and cellular gene expression

Exploring conformational variability of an rna domain in the ribosome: from structure and function to potential antibiotic targeting

RNA in nature is modified at many specific sites to order to gain extra functions or to expand the genetic code. One of such RNAs is ribosomal RNA (rRNA), which contains several modified bases, particularly around the functionally significant sites. We have focused on understanding the influences of modified base on RNA structure and function by employing helix 69 (H69), which is a good region to evaluate the roles of modified bases since it contains three pseudouridines in the loop region and exists at the core of the ribosome. Previous model studies using small hairpin H69 showed the conformational differences of H69 loop under different conditions and revealed the significance of modified bases in H69 dynamics. Comparison of crystal structures of ribosomes indicates variable H69 conformations under different conditions. Based on these information, we performed dimethylsulfate (DMS) probing on 50S ribosomal subunits under different pHs, temperatures and Mg2+ concentrations, showing that H69 has a dynamic RNA loop component on the ribosome level as well as the models, and its multiple conformations are dependent on the presence of modified bases. In addition, footprintings in the presence of aminoglycoside antibiotics neomycin and paromomycin also show conformational variability in H69. These results indicate that H69 exists in multiple conformational states, which could be related to the function of the ribosome in the cell