PDBsum entry 3b5a

RNA

PDB id: 3b5a

Contents

DNA/RNA

Ligands

SO4

NCO ×2

Waters ×10

PDB id:

3b5a

Name:

RNA

Title:

Crystal structure of a minimally hinged hairpin ribozyme incorporating a38g mutation with a 2'ome modification at the active site

Structure:

Loop a substrate strand. Chain: a. Engineered: yes. Other_details: 2'5' phosphodiester linkage between a6 and g7. 29-mer loop a and loop b ribozyme strand. Chain: b. Engineered: yes. Other_details: synthetic polyethylene glycol linker at hinge position 14.

Source:

Synthetic: yes. Other_details: solid-phase synthesis at keck institute, yale. Other_details: solid phase chemical synthesis at dharmacon, colorado. Other_details: solid phase chemical synthesis at dharmacon, colorado

Resolution:

2.35Å R-factor: 0.216 R-free: 0.238

Authors:

C.Macelrevey,J.Krucinska,J.E.Wedekind

Key ref:

C.MacElrevey et al. (2008). Structural effects of nucleobase variations at key active site residue Ade38 in the hairpin ribozyme. Rna, 14, 1600-1616. PubMed id: 18596253

Date:

25-Oct-07 Release date: 12-Aug-08

DNA/RNA chains

U-C-C-C-A2M-G-U-C-C-A-C-C-G 13 bases

C-G-G-U-G-A-G-A-A-G-G-G-S9L-G-G-C-A-G-A-G-A-A-A-C-A-C-A-C-G-A 30 bases

U-C-G-U-G-G-U-G-C-A-U-U-A-C-C-U-G-C-C 19 bases

Rna 14:1600-1616 (2008)

PubMed id: 18596253

Structural effects of nucleobase variations at key active site residue Ade38 in the hairpin ribozyme.

C.MacElrevey, J.D.Salter, J.Krucinska, J.E.Wedekind.

ABSTRACT

The hairpin ribozyme requires functional groups from Ade38 to achieve efficient bond cleavage or ligation. To identify molecular features that contribute to catalysis, structures of position 38 base variants 2,6-diaminopurine (DAP), 2-aminopurine (AP), cytosine (Cyt), and guanine (Gua) were determined between 2.2 and 2.8 A resolution. For each variant, two substrate modifications were compared: (1) a 2'-O-methyl-substituent at Ade-1 was used in lieu of the nucleophile to mimic the precatalytic state, and (2) a 3'-deoxy-2',5'-phosphodiester linkage between Ade-1 and Gua+1 was used to mimic a reaction-intermediate conformation. While the global fold of each variant remained intact, the results revealed the importance of Ade38 N1 and N6 groups. Absence of N6 resulting from AP38 coincided with failure to localize the precatalytic scissile phosphate. Cyt38 severely impaired catalysis in a prior study, and its structures here indicated an anti base conformation that sequesters the imino moiety from the scissile bond. Gua38 was shown to be even more deleterious to activity. Although the precatalytic structure was nominally affected, the reaction-intermediate conformation indicated a severe electrostatic clash between the Gua38 keto oxygen and the pro-Rp oxygen of the scissile bond. Overall, position 38 modifications solved in the presence of 2'-OMe Ade-1 deviated from in-line geometry, whereas variants with a 2',5' linkage exhibited S-turn destabilization, as well as base conformational changes from syn to anti. These findings demonstrate the importance of the Ade38 Watson-Crick face in attaining a reaction-intermediate state and the sensitivity of the RNA fold to restructuring when electrostatic and shape features fail to complement.