RNR brendimas RNR struktūra

Transcription

1 RR brendimas RR struktūra E.coli rrr brendimo schema. Rnazės III taikiniai 1

2 RR antrinės struktūros elementai Tetrakilpos (tetraloops) Tetrakilpos- receptoriaus sąveika Stabilios tetranukleotidinės struktūros rrr (kamienų galuose): GRA > UCG > CUUG GRA GAAA tetraloop 2

3 Pseudomazgas (pseudoknot) Kilpų bučiniai (Kissing loops) (transactivation response element) -formuoja tretinę RR struktūrą -paplitęs virusų sąveikos mechanizmas 3

4 Structural similarities between a kissing complex and a pseudoknot kissing complex pseudoknot -CH 3 -CH= -CH 2 H -CH 2 CH 2 -CH 2 CH -CH 2 CCH 3 -CH(H)CH -CH(H)CCH 3 -CHH 2 -CHH 2 CH 3 -CH 2 HCH 2 CH -H -CH 3 -CH 2 CH -CH 2 CCH 3 Covalent modifications in mature RA C 4 C 5 3 C 6 C 2 H 2 (D) 1 Y = S -HCH 3 -HCCH 3 2 H Pyrimidines derivatives -CH 3 -(CH 2 ) 3 CH(H 2 )CH = S = Se -H(CH 2 ) 4 CH(H 2 )CH -CH 3 = (Inosine) -HCH 3 -(CH 3 ) 2 -HCH 2 CH=C(CH 3 ) 2 -HCH 2 CH=C(CH 3 )CH 2 H -HCHCH 2 CH -HCHCH(CH)CH(H)CH 3 -HCHCH(CH)CH(H)CH 2 CH 3 -CH 3 ucl. Acids Res. 2000, 28 (17): The only atom that has not yet been found modified in any RA analysed so far Total:about 100 distinct modified nucleosides identified so far in all kinds of RA (2004) -CH 3 -CH 2 H 2 (preq1) -C= (preqo) -(H 2 )=H (Q+) -Queuosine (Q) -epoxyqueuosine (oq) -Gal-Queuosine -Man-Queuosine C H -Ribose-P 4 H 2 -CH 3 C C 2 -CH 3 Hypermodification (img,hyw,yw,o2yw) -HCH 3 -(CH 3 ) 2 -SCH 3 Purine derivatives 4

5 RA MDIFICATI Groups addition RA polymer Ribosylation H 2 All blue spots correspond to methylation sites H G - -P= Thiolation H Group addition H 2 - -P= Transglycosylation A Group addition The phosphodiester bond is never broken H Selenation H 2 H - -P= Groups addition Deamination C - -P=0 H H Acetylation Thiolation U RA polymer Formylation Hydroxymethylation Group addition Reduction Isomerisation Motorin & Grosjean et al, 1998 Among cellular RAs, tra is by far the most diversely modified THE WRLD F RA MDIFICATI DISTICT MDIFIED UCLESIDES RA type i 6 A ms 2 i 6 A io 6 A ms 2 io 6 A ms 2 m 6 A Ar(p) Gr(p) Content Diversity sn(o)ra gq preq 0 preq 1 g 6 A t 6 A m 6 t 6 A ms 2 t 6 A hn 6 A ms 2 hn 6 A m 2 Gm m 2 2 Gm Q oq galq manq tra 75-95nt 4-26% mra m 3 Um m 6 2 Am m 2,7 G m 6 Am m2,2,7 G Gm Cm Um Am m 7 G m 5 C C-->U I m 1 Im img mimg HyW* yw HyW o 2 yw k 2 C s 2 C f 5 C f 5 Cm m 5 Cm sn(o)ra nt m 6 A Ym m 2 G Y nm 5 s 2 U mnm 5 U mnm 5 s 2 U mnm 5 se 2 U cmnm 5 U cmnm 5 Um cmnm 5 s 2 U rra mra nt nt 3-20 nt 0-13 nt nt 0-5 nt 0-10% m 1 acp 3 Y D m 3 Y m 1 G m 2 2 G m 5 Um m 5 s 2 U ho 5 U mo 5 U cmo 5 U mcmo 5 U 1-3% <1% m 1 A m 2 A m 4 C m 4 Cm hm 5 C m 3 C ac 4 C ac 4 Cm m 3 U cm 5 U m 1 Y m 5 U ncm 5 U mcm 5 U ncm 5 Um mcm 5 Um chm 5 U mcm 5 s 2 U mchm 5 U S 2 U s 4 U tra rra m 6 2 A Im s 4 U ms 12 UI s 2 Um acp 3 U May ov

6 3D-core structure Examples of simple modifications H H B A S E R i b - m e t h y l C H Anticodon branch 2 H H Complex modifications 5 m 5 U = T C H 3 C H 3 m 1 I n o s i n e S 4 H 2 5 m 5 C S 4 U H 5 2 S C H 3 H 2 m 7 G C H 2 C C H 3 m c m 5 s 2 U H 2 H H H 5 H D 6 H H C H C H 3 H H H 6 2 H 3 C S H 2 H 1 H 5 Y H C H 3 C H H C H C H m s 2 t 6 A k 2 C They are mostly located in functional regions (t+r+snras...) Methylases Isomerases Deaminases Thiolases Acetylases Formylases xydases Reductases Glycosylases Ribosylases Transferases + many others Modified nucleotides in tras are catalysed by a large family of enzymes break C 8 m 1 A m 2 A m 6 A m 6 2A A->I m 1 I i 6 A ms 2 i 6 A g 6 A t 6 A ms 2 t 6 A ms 2 n 6 A 7 9 C C 2 m 1 G m 2 G m 2 2G m 7 G m 2,7 G m 2,2,7 G gq,oq preq GluQ,galQ manq mimg yw(m 3 ) break m 3 C m 4 C m 5 C hm 5 C s 2 C k 2 C f 5 C ac 4 C U->C C 4 C 5 3 C 6 1 C 2 C H 2 m 3 U,m 5 U(T) s 2 m 5 U s 2 U, s 4 U acp 3 U mo 5 U,ho 5 U cm 5 U,mcm 5 U cmo 5 U,mcmo 5 U mnm 5 U,cmnm 5 U mnm 5 s 2 U D, m 5 D - Y m 3 Y m 1 Y m 1 acp 3 Y H P B a s e Am Um Cm Gm Im Ym Ar(p) Gr(P) 6

7 trr struktūra Gm18-s 2 T54 : stabilises 3Dstructure = always present in hyperthermo- Phic organisms D20: increases flexibility of D-loop = present in psychro+mesophiles= absent in thermophiles Structural role tra/protein interaction Translational role Ar(P)64: on tra Met initiator: antideterminant for elongation factor m 1 A9: avoid misfolding Y39: stabilises anticodon branch improves translation efficiency m 1 G37: on tra Asp : antideterminant for Arg-RS translation fidelity ms 2 i 6 A37: stabilises anticodon loop & C/AC inter k 2 C34: on tra Ile : antideterminant for Met-RS pairs to A instead of G U*34: I34: restricts decoding capacity of U or A 7

8 Bazių sąveika trr (A) Distribution of modified residues in tra sequences from E. coli as presented by the tramodviz server. Percent of modified residues within each position is mapped onto the consensus tra secondary structure. (B) Frequency of A, U, C or G bases occurrence and their derivatives in tra sequences from E. coli. (C) Color pie charts which present detailed information for chosen position in tra sorted by the originating base, sorted by the presence of modification or covering modifications only. RA Biology (2014) 11:12, ; 8

9 trr antikodono sritis Grandinės sūkiai RR 9

10 Kodono-antikodono sąveika Genetinis kodas Anticodon (1) I Codon (3) T, C, or A U* A or G G C A 2sU C or U G U A 10

11 Anticodon (1) Codon (3) Bazių poravimasis kodono G 3 C pozicijoje or U I T, C, or A U* A or G C A 2sU G U A Ribosomos struktūra The tertiary structure of the RA in the H. marismortui large ribosomal subunit and its domains. The RA structure of the entire subunit. (A) The subunit particle in its crown view. (B) The crown rotated by 180 about a vertical axis in the plane of the image.. Ban, P. issen, J. Hansen, P. B. Moore, T. A. Steitz, Science 289, 905 (2000) 11

12 Transliacija Ribosomos struktūra The structure of the RA in the H. marismortui large ribosomal subunit and its domains. (C) Schematic secondary structure diagram of 23S rra with the domain coloring used throughout the figures and the helices numbered according to Leffers et al. (D) The secondary structure of 5S rra from H. marismortui. Bases joined by thick lines represent Watson-Crick pairing, and those joined by a lower case "o" indicate non-watson-crick pairing. Bases joined by thin lines interact via a single hydrogen bond, whereas those in black are unpaired. Base pairings shown in red are phylogenetically predicted pairings that are now confirmed. Pairs shown in blue were observed but were not predicted, and pairs shown in green were predicted but were not observed. Ban, P. issen, J. Hansen, P. B. Moore, T. A. Steitz, Science 289, 905 (2000) 12

13 Peptidinės jungties formavimas ribosomoje Ribozyme Ribonucleotide enzyme 1989 obelio premija Chemija Sid Altman Tom Cech 13

14 Ribozimų katalitiniai centrai rrr brendimas savaiminio intronų išsikirpimo (self-splicing) būdu Tetrahymena thermophila rrr brendimo etapai (group I intron self-splicing). (T.R. Cech et al., ature, 1983, 301: 578) 14

15 irr brendimas savaiminio intronų išsikirpimo būdu. (group II introns) SELEX DR SELEX Pradinė kombinatorinė biblioteka (viengrandė DR) Fiksuota Fiksuota seka Kombinatorinė sritis seka (18 21 nt) (20 80 nt) (18 21 nt) T7 promotorius 1 2 atv. PGR Dvigrandės DR biblioteka atv. 5 Systematic Evolution of Ligands by Exponential enrichment Kombinatorinė RR biblioteka 5 T7 RR polimerazė (transkripcija in vitro) 3 RR SELEX 15

16 SELEX 16