PDBsum entry 1hi0

RNA polymerase

PDB id: 1hi0

Contents

Protein chains

664 a.a.

DNA/RNA

Ligands

GTP ×6

Metals

_MG ×6

_MN ×3

PDB id:

1hi0

Name:

RNA polymerase

Title:

RNA dependent RNA polymerase from dsrna bacteriophage phi6 plus initiation complex

Structure:

DNA (5'-( Tp Tp Tp Cp C)-3'). Chain: d, e, f. Engineered: yes. Other_details: 5 nucleotide DNA version of optimum RNA template. P2 protein. Chain: p, q, r. Synonym: RNA-directed RNA polymerase. Engineered: yes

Source:

Synthetic: yes. Bacteriophage phi-6. Phage phi 6. Organism_taxid: 10879. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Hetero-Dimer (from PDB file)

Resolution:

3.00Å R-factor: 0.214 R-free: 0.239

Authors:

J.M.Grimes,S.J.Butcher,E.V.Makeyev,D.H.Bamford,D.I.Stuart

Key ref:

S.J.Butcher et al. (2001). A mechanism for initiating RNA-dependent RNA polymerization. Nature, 410, 235-240. PubMed id: 11242087 DOI: 10.1038/35065653

Date:

31-Dec-00 Release date: 27-Mar-01

Protein chains

P11124  (RDRP_BPPH6) -  RNA-directed RNA polymerase from Pseudomonas phage phi6

Seq: 665 a.a.

Struc: 664 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

T-T-C-C 4 bases

T-T-C-C 4 bases

T-T-C-C 4 bases

Enzyme reactions

• Enzyme class: E.C.2.7.7.48 - RNA-directed Rna polymerase.
• Reaction: RNA(n) + a ribonucleoside 5'-triphosphate = RNA(n+1) + diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1038/35065653

Nature 410:235-240 (2001)

PubMed id: 11242087

A mechanism for initiating RNA-dependent RNA polymerization.

S.J.Butcher, J.M.Grimes, E.V.Makeyev, D.H.Bamford, D.I.Stuart.

ABSTRACT

In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail. Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.

Selected figure(s)

Figure 2.
Figure 2: Key aspects of various phi-6 polymerase structures. a, 6 polymerase sliced open; arrows highlight key features. The areas of the surrounding close-ups in b-e are marked by semitransparent boxes, and orientations shown are with respect to a. b, Surface representation28 viewed from above showing the entrance to the template tunnel. Putative positions for the strands before initiation are shown. c, A section through the template channel with the bound oligomer drawn in yellow. The surface of the polymerase and the embedded polypeptide chain are coloured green. The two 3' cytidines are marked as T1 and T2. d, Difference electron density map for the NTP bound to site I (based on ninefold averaging of three difference density maps). ATP is drawn with the phosphates coloured green. e, Stereo image of the initiation complex. The 3' cytidines (T1 & T2) are drawn in blue. The incoming GTPs, D1 and D2, are shown base paired (bonds in red) to T1 and T2. Y630 ring stacks with the base of D1. D453, D454 and D324 are the catalytic aspartates, the Mn2+ ion is shown in cyan, the two catalytic Mg2+ ions are in green.

Figure 3.
Figure 3: Models for initiation and chain elongation. a Cartoon illustrating key points in the reaction mechanism for phi-6 polymerase. Red boxes highlight experimental results. I, apo structure with bound Mn2+. Binding sites are identified in black letters. II, NTP bound in site I. II|I, template bound. IV, template bound and NTP non-productively bound at site I. V, initial productive binding at site P. VI, template ratchets back. VII, second GTP bound at site P. Polymerization can occur. VIII, polymerization has occurred, releasing nascent duplex from ordered binding at the active site C. The C-terminal domain moves allowing the duplex to ratchet forward, out of the active site. b, 6 polymerase and polymerases of the Reoviridae family in the context of the viral capsid. Polymerases are coloured yellow. In the Reoviridae panel the helicase is orange, and the 5' end of the positive strand is attached to the polymerase, holding the genome segment, ready to facilitate re-initiation.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2001, 410, 235-240) copyright 2001.

Figures were selected by an automated process.