Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment

Abstract

All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3 0 ,5 0 -phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA -protein world.

Crystal structure determination of anti‐DNA Fab A52

“…Fabs 64M‐2 and 64M‐5 are specific for UV‐irradiated/damaged DNA, specifically the DNA (6‐4) photoproduct, and 64M‐2 has been crystallized with a small photoproduct, while the 64M‐5 has recently been reported in complex with a large double stranded DNA containing the flipped out bases of the photoproduct that interact with the Fab . Jel103, a Fab specific for single‐stranded RNA has been crystallized in the unliganded form and with single RNA bases, while structures for Fabs Fab2 and BL3–6 in complex with large ribozyme domains have also been determined . Like A52, Fabs BV04‐01, DNA‐1, and 3D8 are anti‐DNA antibodies derived from SLE‐prone mice, while the other antibodies were generated by immunization of mice with DNA–protein complex, UV‐irradiated DNA, mixed DNA–RNA complexes, or selected from synthetic phage display libraries.…”

“…All the Fabs are derived from mice, and all the light chains are of the kappa class. With the exception of BV04‐01, all of the heavy chains derive from the VH1 family, with sequence identity to A52 heavy chain ranging from 44 to 78%, and all the CDR H3 regions have at least one arginine residue, with as many as 4 (for A52) and 5 (for BL3–6, isolated from a phage‐display library enriched for Tyr, Ser, Gly and Arg in CDRs). A52 is reported to bind both single‐ and double stranded DNA, so that a comparison with Fabs bound to both of these species is of interest.…”

Mapping L1 Ligase Ribozyme Conformational Switch

“…We can draw parallels between L1L conformational transition and presumed catalytic mechanism of other ribozymes, such as hammerhead ribozyme (HHR) and the recently crystallized class I RNA polymerase (CIRP)[92, 114]. Both HHR and L1L have an active and inactive states but the sequences and structures are completely different, and the conformational changes they undergo are also unique.…”

“…Both ribozymes X-ray structures have a Mg 2+ ion resolved near the catalytic site. For both ribozymes an additional Mg 2+ has been proposed to stabilize a triphosphate group in the active site as suggested by analysis of CIRP crystal structure [92, 114] and theoretical investigations on L1L and CIRP[48, 113] These findings fit in a much larger context of DNA and RNA polymerases, nucleases and transposases for which the proximity of two Mg 2+ to the active site has been proposed to enhance substrate recognition and catalytic specificity[126]. …”

The chemical origins of life and its early evolution: an introduction

“…Clearly, a fundamental requirement would be a ribozyme that can replicate RNA, and the related ligase, and these have been isolated in the Bartel laboratory [16,17]. Piccirilli & Koldobskaya [18] (Chicago) present the structure of the Bartel ligase RNA, arguing for a two-metal ion mechanism. Would such ribozymes have been efficient enough to support self-sustaining RNA-based organisms?…”