Bakteri ribosoomide uurimus keemilise modifitseerimise meetoditega

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Ribosoom on suur makromolekulaarne kompleks, mis kodeerib päriliku informatsiooni valgulisse olemusse. Eeltuumsete organsimide ribosoom koosneb kaheks alamühikust, väikesest (30S) ja suurest (50S) alamühikust. Ribosoomi kahte alamühikut hoiavad koos ~ 30 erinevat ühendust, mis on jagatud 12 silla (B1a-B8) vahel. Väike alamühik koosneb ühest RNA molekulist (16S rRNA, 1542 nukleotiidi) ja 21-st ribosoomi valgust (S1-S21). Ribosoomi suur alamühik koosneb kahest RNA molekulist (5S rRNA, 120 nukleotiidi ja 23S rRNA, 2904 nukleotiidi) ja 33-st ribosoomi valgust (L1-L36). Minu töös uuritakse ribosomaalse RNA keemiliste positsioonide olulisust ribosoomi kahe alamühiku omavahelisel seonumisel. Keemilise modifitseerimise meetodit kasutades detekteerisime 16S rRNA-s kuus positsiooni (A702, A1418, A1483, U793, U1414 ja U1495), millede modifitseerimine takistab alamühikute assotseerumist. Detekteeritud positsioonid paiknevad tuntud alamühikute vahelistes sildades. Seega alamühikute assotsiatsioonil mängivad olulist rolli sillad B2a (U1495), B2b(U793), B3 (A1418, A1483, U1414) ja B7a (A702). Lisaks sellele töötasime välja meetodi, millega saab uurida RNA suhkur-fosfaat selgroo interaktsioone 23S rRNA-s. Välja töötatud meetodit on võimalik kasutada RNA suhkur-fosfaat selgroo interaktsioonide uurimiseks, substraatide sidumiskohtade määramiseks ja individuaalsete positsioonide mõju määramiseks valgusünteesi erinevates etappides. Kolmandas töös uuritakse ribosoomi valkude võimet välja vahetuda ja selle tulemusena taastada keemiliselt kahjustatud ribosoomide funktsioon. Ribosoomis välja vahetuvate valkude kindlaks tegemiseks, me kasutasime kahte in vitro meetodit, nii radioaktiivset märgistamist kui ka raskete isotoopide eristamise meetodit. Ribosoomi valgud S2, L1, L7/12, L9, L10, L11 ja L33 on kõige kergemini vahetuvad r-valgud. Seega, meie tulemused näitavad, et kahjustatud ribosoome on võimalik parandada valkude asendamise teel.The ribosome is a macromolecular assembly that is responsible for protein biosynthesis following genetic instructions in all organisms. The prokaryotic ribosome contains about two-thirds RNA and one-third protein and consists of two subunits, the larger (50S) of which is approximately twice the molecular weight of the smaller (30S). Prokaryotic ribosomes contain ~54 different proteins, 23S rRNA, 16S rRNA, and 5S rRNA. Two ribosomal subunits are held together by more than 30 individual intersubunit interactions spread among 12 bridges (B1-B8). Using modification interference approach we were able to identify 6 essential 16S rRNA positions for subunit association. Modification of the N1 position of A702, A1418, and A1483 with DMS, and of the N3 position of U793, U1414, and U1495 with CMCT in 30S subunits strongly interferes with 70S ribosome formation. Five of these positions localize into previously recognized intersubunit bridges, namely, B2a (U1495), B2b (U793), B3 (A1483; A1418), and B7a (A702). These four intersubunit bridges are essential for reassociation of the 70S ribosome, thus forming the functional core of the intersubunit contacts. In order to study RNA backbone interactions in the ribosome, we combined different assays like in vitro T7 transcription, in vitro 50S reconstitution and primer extension to generate a reliable approach to study RNA backbone interactions of the large ribosomal subunit by using phosphorothioate approach. This phosphorothioate-substitution approach is suitable for footprinting of various ligand-ribosome complexes and for functional studies in the modification interference assay. In addition, because the ribosome is made of many individual proteins, we studied the ability of ribosomal proteins to exchange and restore the function of damaged ribosomes. Incubation of chemically inactivated ribosomes with total ribosomal proteins led to reactivation of translational activity. Ribosomal proteins S1, S2, L1, L7/12, L9, L10, L11 and L33 are among the most readily exchangeable proteins. The results show that the damaged ribosomes can be repaired by mean of protein exchange

SecM-Stalled Ribosomes Adopt an Altered Geometry at the Peptidyl Transferase Center

A structure of a ribosome stalled during translation of the SecM peptide provides insight into the mechanism by which the large subunit active site is inactivated

The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation

Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the only sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlAcrb pause peptide

Structural studies of ribosome stalling and translocation complexes

In this study, cryo-electron microscopy (cryo-EM) and single particle reconstruction were used as a main technique to investigate the involvement of bacterial ribosomes in two crucial cellular processes: the regulation of gene expression and the biogenesis of membrane proteins. Whereas most nascent chains are thought to transit passively through the ribosomal exit tunnel during translation, a number of regulatory peptide sequences, such as TnaC and SecM, have been proposed to specifically interact with tunnel components, causing the ribosome to stall which in turn regulates the expression of downstream gene products. In the first part of this study, a 5.8 Å resolution cryo-EM reconstruction of an Escherichia coli 70S ribosome stalled during translation of the TnaC leader peptide could be determined. The high quality of the map allowed the visualization of the TnaC nascent chain within the exit tunnel of the ribosome, making contacts with ribosomal components at distinct sites. At the peptidyl transferase center (PTC), the universally conserved nucleotides A2602 and U2585 adopt conformations that are incompatible with co-habitation of the termination release factors. Moreover, a model could be proposed where interactions within the tunnel are relayed back to the PTC, leading to its inactivation. In addition, a foundation for the elucidation of the SecM-stalling mechanism could also be established. The membrane protein insertase YidC is the prokaryotic member of the conserved YidC/Oxa1/Alb3 protein family. It assists in the assembly and folding of membrane proteins in conjunction with the Sec translocase as well as on its own. E. coli YidC is a hexaspan protein with a large, non-conserved periplasmic domain between the first and second transmembrane (TM) segment. In contrast, YidC2 from the Gram-positive bacterium Streptococcus mutans contains five TM segments and an extended C-terminal region akin to the C-terminal ribosome binding domain of the mitochondrial YidC homolog Oxa1. In the second part of this study, programmed 70S ribosomes carrying the YidC-specific nascent chain MscL could be generated, and visualized in a preliminary low-resolution cryo-EM structure in complex with E. coli YidC. Furthermore, purified S. mutans YidC2 was reconstituted into proteoliposomes and the formation of a ribosome-YidC2-proteoliposome complex could be demonstrated. Thus, the foundations have been laid for the visualization of YidC2 in the membrane environment. Improvement of the preliminary RNC-YidC structure together with determination of an RNC-YidC2 complex are expected to provide insights into the molecular mechanism of YidC mediated membrane protein biogenesis

Ribosome Structure and the Mechanism of Translation

AbstractThe publication of crystal structures of the 50S and 30S ribosomal subunits and the intact 70S ribosome is revolutionizing our understanding of protein synthesis. This review is an attempt to correlate the structures with biochemical and genetic data to identify the gaps and limits in our current knowledge of the mechanisms involved in translation

Tuning ribosomal elongation cycle by mutagenesis of 23S rRNA

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

Structural analysis of stalled ribosomal complexes and their respective rescue mechanisms by Cryo-Electron Microscopy

The ribosome is a multifunctional ribonucleoprotein complex responsible for the translation of the genetic code into proteins. It consists of two subunits, the small ribosomal subunit and the large ribosomal subunit. During initiation of translation, both subunits join and form a functional 70S ribosome that is capable of protein synthesis. In the course of elongation, the ribosome synthesizes proteins according to the codons on the mRNA until it encounters a stop codon leading to the recruitment of release factors 1 or 2 followed by release of the nascent chain. Upon release of the polypeptide chain the subunits dissociate from each other and can be recruited for another round of translation. There are two scenarios that interfere with active translation, namely the formation of so called ‘non-stop’ or ‘no-go’ complexes. In both cases, ribosomes pause translation and without interference of additional factors, they would become stalled. Accumulation of such events leads to a decrease of ribosomal subunits that can be recruited for translation, ultimately resulting in the death of the cell. Using cryo-electron microscopy (cryo-EM), we obtained the structure of alternative rescue factor A (ArfA) together with release factor 2 bound to a ‘non-stop’ complex. Our reconstructions showed that the C-terminal domain of ArfA occupies the empty mRNA channel on the SSU, whereas the N-terminal domain provides a platform for recruiting RF2 in a stop codon-independent way. Thereby, ArfA stabilizes a unique conformation of the switch loop of RF2, responsible for directing the catalytically important GGQ motif towards the PTC. The high-resolution structure of ArfA allowed us to compare its mode of action with trans-translation and alternative rescue factor B, two other factors operating on ‘non-stop’ complexes. A second project focused on elongation factor P (EF-P), a factor that alleviates stalling on polyproline stalled ribosomes. Applying cryo-EM, we were able to show that in the absence of EF-P, the nascent chain is destabilized as the polyproline moiety attached to the P-tRNA is not able to accommodate within the ribosomal tunnel. Binding of modified EF-P to the polyproline stalled complex stabilizes the P-site tRNA and especially the CCA, thereby forcing the nascent chain to adopt an alternative conformation that is favorable for translation to proceed