PDBsum entry 1i6j

Transferase/DNA

PDB id: 1i6j

Contents

Protein chain

256 a.a. *

DNA/RNA

Waters ×204

* Residue conservation analysis

PDB id:

1i6j

Name:

Transferase/DNA

Title:

Crystal structure of a pseudo-16-mer DNA with stacked guanines and two g-a mispairs complexed with the n-terminal fragment of moloney murine leukemia virus reverse transcriptase

Structure:

5'-d( Gp Tp Cp Gp Tp C)-3'. Chain: b. Engineered: yes. 5'-d( Ap Cp Gp Gp Gp Ap Cp Gp Ap C)-3'. Chain: c. Engineered: yes. Reverse transcriptase. Chain: a. Fragment: n-terminal fragment.

Source:

Synthetic: yes. Other_details: abi 392 DNA synthesizer. Moloney murine leukemia virus. Organism_taxid: 11801. Gene: virus. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Biol. unit:

Hexamer (from PQS)

Resolution:

2.00Å R-factor: 0.225 R-free: 0.252

Authors:

M.L.Cote,M.M.Georgiadis

Key ref:

M.L.Coté and M.M.Georgiadis (2001). Structure of a pseudo-16-mer DNA with stacked guanines and two G-A mispairs complexed with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase. Acta Crystallogr D Biol Crystallogr, 57, 1238-1250. PubMed id: 11526315 DOI: 10.1107/S090744490100943X

Date:

02-Mar-01 Release date: 07-Sep-01

Protein chain

P03355  (POL_MLVMS) -  Gag-Pol polyprotein from Moloney murine leukemia virus (isolate Shinnick)

Seq: 1738 a.a.

Struc: 256 a.a.*

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

DNA/RNA chains

G-T-C-G-T-C 6 bases

A-C-G-G-G-A-C-G-A-C 10 bases

Enzyme reactions

• Enzyme class 2: E.C.2.7.7.- - ?????
• Enzyme class 3: E.C.2.7.7.49 - RNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
• Enzyme class 4: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
• Enzyme class 5: E.C.3.1.-.-
• Enzyme class 6: E.C.3.1.26.4 - ribonuclease H.
• Reaction: Endonucleolytic cleavage to 5'-phosphomonoester.
• Enzyme class 7: E.C.3.4.23.- - ?????

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

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

reference

DOI no: 10.1107/S090744490100943X

Acta Crystallogr D Biol Crystallogr 57:1238-1250 (2001)

PubMed id: 11526315

Structure of a pseudo-16-mer DNA with stacked guanines and two G-A mispairs complexed with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase.

M.L.Coté, M.M.Georgiadis.

ABSTRACT

The X-ray crystal structure at 2.0 A resolution of a DNA molecule complexed with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase (MMLV RT) has been determined. This method allows the study of nucleic acids in a unique and largely unfettered environment without the complicated lattice interactions typically observed in DNA-only crystal structures. Molecular-replacement phasing using only the protein provided readily interpretable electron density with no model bias for the DNA. The asymmetric unit of the structure consists of the protein molecule bound to the blunt end of a DNA 6/10-mer, which is composed of a six-base strand (5'-GTCGTC-3') and a ten-base strand (3'-CAGCAGGGCA-5'), resulting in a six-base-pair duplex with a four-base single-stranded overhang. In the crystal structure, the bases of the overhang reciprocally pair to yield a doubly nicked pseudo-hexadecamer primarily B-form DNA molecule. The pairing between the single strands gives two standard (G-C) Watson-Crick pairs and two G(anti)-A(anti) mispairs. The mispairs reside in a G-C-rich environment and the three consecutive guanines on the 10-mer impart interesting structural features to the pseudo-hexadecamer, such as the preference for a guanine stack, stretching the C-G base pairs flanking the mispair to the point of loss of intra-base-pair hydrogen bonding. The DNA was designed for the purpose of comparison with a previous structure, which was determined in the same crystal lattice. In all of the authors' previous fragment-DNA complexes, the nucleotide at the blunt-ended 3'-hydroxyl was a purine. Consistent with the proposed mechanistic role of interactions with the 3'-hydroxyl in processive DNA synthesis by RT, it was found that a pyrimidine at this position in the DNA makes indentical interactions with the strictly conserved Gly191 and the main chain of Leu115 of MMLV RT.

Selected figure(s)

Figure 4.
Figure 4 Comparative views (Kraulis, 1991[Kraulis, P. J. (1991). J. Appl. Cryst. 24, 946-950.]; Merritt & Bacon, 1997[Merritt, E. A. & Bacon, D. J. (1997). Methods Enzymol. 277, 505-524.]) of the protein-DNA binding sites of the (a) form IVa and the (b) form IVb structures. In each view, the characteristic ion-pair between Asp114 and Arg116 is shown with black dotted lines. Green dotted lines denote hydrogen bonds whose distances range from 2.4 to 3.3 Å. Magenta dashed lines represent contacts whose distances are greater than 3.3 Å and less than 3.8 Å. Note the difference in the disposition of the Asp114-Arg116 ion pair in its interaction with the nucleic acid in the form IVa versus the IVb structure. Note the absence of contacts to the DNA from Tyr64 in the form IVb structure.

Figure 6.
Figure 6 Stereoview (Kraulis, 1991[Kraulis, P. J. (1991). J. Appl. Cryst. 24, 946-950.]; Merritt & Bacon, 1997[Merritt, E. A. & Bacon, D. J. (1997). Methods Enzymol. 277, 505-524.]) of the form IVa and form IVb pseudo-hexadecamers resulting from the superpositioning of the C^ atoms of the protein molecules of their structures. Note the near-exact match of the 3'-OH ribose rings and the lack of matches elsewhere. The form IVb DNA is shown in red and the form IVa DNA is shown in white, retaining its A7 base in the anti conformation.

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2001, 57, 1238-1250) copyright 2001.

Figures were selected by an automated process.