Studies on bacterial ribosomes by chemical modification approaches
Date
2010-07-29
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Abstract
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.
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.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
doktoritööd, molekulaarbioloogia, bakterid, ribosoomid, keemilise modifitseerimise meetodid