Nucleic acid structure Microreview 1 er : sequence of the nucleotides, each distinguished by its base. 2 ary : antiparallel double helix. 3 ary : folding of sequentially remote secondary structure to form 3-d entity: coaxial stacks, pseudoknots, ribose zippers, cation-binding sites. 4 ary : association of multiple molecules to form a noncovalent complex, often in association with proteins. 1
Tertiary structure of RNA Coaxial stacks of bases from the bases of different stem-loops. Pseudoknots: H-bonding between loop bases and nearby SS-RNA. Ribose zippers: H-bonds between 2 OH groups of ribose in antiparallel SS strands. Mg 2+ binding, NEXT. Three-base H-bonding. Fig. 11.34 of Garrett & Grisham 2
Translation: trna and Ribosomes 1-6 Strong sites: Kd~10-5 M, cooperative. Weak sites, statistical, ionic strength effects. Holbrook et al (1977) NAR 4(8) 2811. 3
Higher-order DNA structure & K + ions. G-quartets in DNA Kondo et al. (2004) NAR 32(8) 2541. Different binding sites depending on K + : DNA ratio. Also: see Thermodynamics of RNA folding vs. [Mg 2+ ]. 4
Quaternary structure of RNA 2 er Str. 3 er and 4 er structure 50s subunit of ribosome including 5S rrna and 23S rrna. RNA accounts for 65% of the mass of this particle. 5 Fig. 11.37 of Garrett & Grisham
Central dogma Replication, transcription, translation (the simple version). DNA mrna Protein rrna, trna Replication: DNA + dntp 2 DNA DNA polymerase (Mg 2+ ) Transcription: DNA + NTP DNA + RNA RNA polymerase (Mg 2+ ) Translation: RNA + aa RNA + protein ribosomes (Mg 2+ ) 6 mrna, trna, rrna are involved
mrna from prokaryotes vs. eukaryotes. 7 G&G Fig. 10.24
Transcription: RNA polymerase Mg 2+ and Zn 2+ 8 Fig3, Cramer (2000) Science 288:640
mrna from prokaryotes vs. eukaryotes. Protein synthesis video, Stanford U. in the early 70s. search Paul Berg and protein synthesis. 9 G&G Fig. 10.24
The ribosome MW 2.7 x 10 6 Da. (E. coli) Crystal structure of a Thermus thermophilus 70S ribosome containing three bound transfer RNAs (top) and exploded views showing its different molecular components (middle and bottom). The 16S, 23S, and 5S ribosomal RNAs are cyan, gray, and light purple, respectively; the A-, P-, and E-site transfer RNAs are shown in yellow, orange, and red, respectively. The 30S subunit proteins are dark purple, and the 50S proteins are magenta. A, P, E 30S 16S 5S 23S 50S 10
The ribosome 11
Translation: trna and Ribosomes trna molecules serve as adaptors basepairing with one codon on the mrna and bringing along one amino acid for the growing peptide chain. Acceptor, Peptidyl and Exit sites. 12 Figs. 30.19 of G&G
Formation of new peptide linkages 13 Figs. 30.21 of G&G
Charging of trna Errors only 1 in 10 4-10 5 1 in 10 2-10 3 x, Ser is adenylated, but it is hydrolize at the acylation site. 14 Figs. 30.3 of G&G Accuracy: For Thr vs. Val, Ser recognition, a Zn 2+ binds OH, not CH3 of Val.
Mg 2+ in the 30s ribosomal fragment James M. Ogle, et al. (2001) Science 292: pp 897-902. Recognition of Cognate Transfer RNA by the 30 Ribosomal Subunit. 15
70s Ribosomal fragment (2006) Science 313 1935-1943 16
Mg 2+ bridges 45 kink in mrna between P and A sites, also binds bkbn Phosphates of Helix44 residues 1400,1401. 3Fo-2Fc density is outlined. All green balls are Mg 2+. Selmer (2006) Science 313 1935-1943. 17
Mg ions in 70s ribosomal subunit. In the 70s Ribosome, numerous Mg 2+ ions are implicated in bridges between ribosomal fragments. Interactions with the RNA most commonly exploit backbone phosphate (non-bridging O). This is proposed to accommodate the shifts in trna vs. mrna and rrna and protein required as part of ribosome function, as the trnas shift from the A site to the P site. Selmer (2006) Science 313 1935-1943. 18
Bridges between domains and subunits. C: Mg links the 2ʼ-OH of C770 and O2 of C899 of 16S, as well as non-bridging phosphate Os of 23S. D: L14 protein and 16S and 23S are bridged by Mg bound to side chains of Glu and non-bridgin phosphate Os. E:L14 and L19 with 16S RNA, backbone oxygens of both Val115 and Ala118 of BUT: side chain of Lys35 of L19 and h14 of 16S RNA 19 Selmer (2006) Science 313 1935-1943.