Transcription-translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits.

In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation

Spatial organization of bacterial transcription and translation

In bacteria such as $\textit{Escherichia coli}$, DNA is compacted into a nucleoid near the cell center, while ribosomes$-$molecular complexes that translate messenger RNAs (mRNAs) into proteins$-$are mainly localized at the poles. We study the impact of this spatial organization using a minimal reaction-diffusion model for the cellular transcriptional-translational machinery. Our model predicts that $\sim 90\%$ of mRNAs are segregated to the poles and reveals a "circulation" of ribosomes driven by the flux of mRNAs, from synthesis in the nucleoid to degradation at the poles. To address the existence of non-specific, transient interactions between ribosomes and mRNAs, we developed a novel method to efficiently incorporate such transient interactions into reaction-diffusion equations, which allowed us to quantify the biological implications of such non-specific interactions, e.g. for ribosome efficiency

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

Characterization of a 30S Ribsomal Subunit Intermediate Found in \u3cem\u3eEscherichia coli\u3cem\u3e Cells Growing with Neomycin and Paromomycin.

The bacterial ribosome is a target for inhibition by numerous antibiotics. Neomycin and paromomycin are aminoglycoside antibiotics that specifically stimulate the misreading of mRNA by binding to the decoding site of 16S rRNA in the 30S ribosomal subunit. Recent work has shown that both antibiotics also inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. This work describes the characteristics of an assembly intermediate produced in E.coli cells grown with neomycin or paromomycin. Antibiotic treatment stimulated the accumulation of a 30S assembly precursor with a sedimentation coefficient of 21S. The particle was able to bind radio labeled antibiotics both in vivo and in vitro. Hybridization experiments showed that the 21S precursor particle contained 16S and 17S rRNA. Ten 30S ribosomal proteins were found in the precursor after inhibition by each drug in vivo. In addition, cell free reconstitution assays generated a 21S particle during incubation with either aminoglycoside. Precursor formation was inhibited with increasing drug concentration. This work examines features of a novel antibiotic target for aminoglycoside and will provide information that is needed for the design of more effective antimicrobial agents

Escherichia coli Ribosoomivalkude süntees statsionaarses kasvufaasis

The repression of cell growth as a response to starvation leads the bacterial culture to enter stationary growth phase. The cell needs to conserve the energy to survive the starvation. As a result, the metabolic processes of the cell slow down. The ribosome is one of the most energy-consuming organelles, as its main function is to carry out the protein synthesis. The ribosomal proteins have a profound effect on the correct functioning and assembly of the ribosome. To determine whether or not the ribosomal proteins of Escherichia coli are synthesised during stationary growth phase, the changes in the protein quantities over 14 days after the beginning of the growth were calculated. 24 out of 54 ribosomal proteins were found to be synthesised during stationary growth phase. Many of these proteins were found to have a positive effect on the cell during stationary growth phase, such as maintenance of the ribosome structural integrity, for instance, via protein exchange, and self-downregulation of the ribosomal protein synthesis. In estonian: Rakkude kasvu aeglustumisel stressitingimustel siseneb rakukultuuri statsionaarsesse kasvufaasi. Selle tulemusena aeglustub rakkude metabolism ning väheneb nende energiakasutus. Ribosoomid on ühed suurimatest energiatarbijatest rakkudes, viies läbi valkude sünteesi ehk translatsiooni. Ribosoomivalgud osalevad ribosoomi tertsiaalstruktuuri korrektses moodustumises ja funktsionaalsuse tagamises. Selleks, et detekteerida Escherichia coli ribosoomivalkude sünteesi statsionaarse kasvufaasi käigus, kasvatati rakke 14 päeva ning mõõdeti valkude koguse muutust rakukultuuris. Tulemused näitasid, et 24 r-valku 54st sünteesiti rakkudes statsionaarse kasvufaasi käigus. Sünteesitud r-valgud võivad omada positiivset efekti rakkudele, alustades ribosoomivalkude sünteesi repressiooniga ning lõpetades olemasolevate ribosoomide struktuuri terviklikkuse hoidmisega, läbi r-valkude vahetuse

Distinct GDP/GTP bound states of the tandem G-domains of EngA regulate ribosome binding

EngA, a unique GTPase containing a KH-domain preceded by two consecutive G-domains, displays distinct nucleotide binding and hydrolysis activities. So far, Escherichia coli EngA is reported to bind the 50S ribosomal subunit in the guanosine-5′-trihosphate (GTP) bound state. Here, for the first time, using mutations that allow isolating the activities of the two G-domains, GD1 and GD2, we show that apart from 50S, EngA also binds the 30S and 70S subunits. We identify that the key requirement for any EngA–ribosome association is GTP binding to GD2. In this state, EngA displays a weak 50S association, which is further stabilized when GD1 too binds GTP. Exchanging bound GTP with guanosine-5′-diphosphate (GDP), at GD1, results in interactions with 50S, 30S and 70S. Therefore, it appears that GD1 employs GTP hydrolysis as a means to regulate the differential specificity of EngA to either 50S alone or to 50S, 30S and 70S subunits. Furthermore, using constructs lacking either GD1 or both GD1 and GD2, we infer that GD1, when bound to GTP and GDP, adopts distinct conformations to mask or unmask the 30S binding site on EngA. Our results suggest a model where distinct nucleotide-bound states of the two G-domains regulate formation of specific EngA–ribosome complexes