PDBsum entry 1kqs

Ribosome

PDB id: 1kqs

Contents

Protein chains

237 a.a. *

337 a.a. *

246 a.a. *

140 a.a. *

172 a.a. *

119 a.a. *

29 a.a. *

156 a.a. *

142 a.a. *

132 a.a. *

145 a.a. *

194 a.a. *

186 a.a. *

115 a.a. *

143 a.a. *

95 a.a. *

150 a.a. *

81 a.a. *

119 a.a. *

53 a.a. *

65 a.a. *

154 a.a. *

82 a.a. *

142 a.a. *

73 a.a. *

56 a.a. *

46 a.a. *

92 a.a. *

DNA/RNA

Ligands

_DC-_DC

PPU-PHA-ACA-BTN

Metals

_CL ×22

_NA ×86

_MG ×117

_CD ×5

__K ×2

Waters ×7871

* Residue conservation analysis

References listed in PDB file

Key reference

Title

A pre-Translocational intermediate in protein synthesis observed in crystals of enzymatically active 50s subunits.

Authors

T.M.Schmeing, A.C.Seila, J.L.Hansen, B.Freeborn, J.K.Soukup, S.A.Scaringe, S.A.Strobel, P.B.Moore, T.A.Steitz.

Ref.

Nat Struct Biol, 2002, 9, 225-230. [DOI no: 10.1038/nsb767]

PubMed id

11828326

Abstract

The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3prime prime or minute OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.

Figure 1.
Figure 1. Schematic of the modified fragment assay. The substrates are shown on the left. CCA-phenylalanine-caproic acid-biotin (CCA-pcb) and C-puromycin (C-pmn) undergo a ribosome-dependent reaction in which a peptide bond is formed between the -amino group of C-pmn and the carbonyl ester of the phenylalanine moiety of CCA-pcb, yielding the two products: C-puromycin-phenylalanine-caproic acid-biotin (C-pmn-pcb) and a deacylated CCA.

Figure 4.
Figure 4. Structure of the new fragment reaction products bound to the ribosome. a, A space-filling representation of the 50S particle (RNA in white and protein in yellow) in complex with products, with the three tRNAs as they were observed^25 binding to the Thermus thermophilus 70S ribosome superimposed for reference. The subunit has been split through the tunnel, and the front half was removed to reveal the tunnel and the peptidyl transferase site (boxed). The orientation is the crown view, with the L1 protein to the left and the L7−L12 stalk to the right. b, A close-up view of the active site shows that the peptidyl-product (CC-Pmn-pcb) (green) binds the A-loop (yellow), whereas the deacylated product (CCA) (violet) base pairs to the P-loop (blue). The N3 of A2486 (A2451) (light blue) is in proximity to the 3' OH of the deacylated product, and the base of U2620 (U2585) (red) has moved near to the newly formed peptidyl ester link and the 3' OH of dimethyl A76.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2002, 9, 225-230) copyright 2002.