PDBsum entry 1po6

RNA binding protein/DNA

PDB id: 1po6

Contents

Protein chain

183 a.a. *

DNA/RNA

Waters ×155

* Residue conservation analysis

PDB id:

1po6

Name:

RNA binding protein/DNA

Title:

Crystal structure of up1 complexed with d(tagg(6mi)ttaggg): a human telomeric repeat containing 6-methyl-8-(2-deoxy-beta-ribofuranosyl) isoxanthopteridine (6mi)

Structure:

5'-d( T Ap Gp Gp (6Mi)p Tp Tp Ap Gp Gp G)-3'. Chain: b. Engineered: yes. Heterogeneous nuclear ribonucleoprotein a1. Chain: a. Fragment: residues 8-190. Synonym: helix-destabilizing protein, single-strand binding protein, hnrnp core protein a1. Engineered: yes

Source:

Synthetic: yes. Other_details: oligonucleotide d(tagg(6mi)ttaggg) based on human telomeric repeat d(ttaggg)n. Homo sapiens. Human. Organism_taxid: 9606. Gene: hnrpa1. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Tetramer (from PQS)

Resolution:

2.10Å R-factor: 0.234 R-free: 0.261

Authors:

J.C.Myers,S.A.Moore,Y.Shamoo

Key ref:

J.C.Myers et al. (2003). Structure-based incorporation of 6-methyl-8-(2-deoxy-beta-ribofuranosyl)isoxanthopteridine into the human telomeric repeat DNA as a probe for UP1 binding and destabilization of G-tetrad structures. J Biol Chem, 278, 42300-42306. PubMed id: 12904298 DOI: 10.1074/jbc.M306147200

Date:

13-Jun-03 Release date: 11-Nov-03

Protein chain

P09651  (ROA1_HUMAN) -  Heterogeneous nuclear ribonucleoprotein A1 from Homo sapiens

Seq: 372 a.a.

Struc: 183 a.a.

DNA/RNA chain

T-A-G-G-6MI-T-T-A-G-G-G 11 bases

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1074/jbc.M306147200

J Biol Chem 278:42300-42306 (2003)

PubMed id: 12904298

Structure-based incorporation of 6-methyl-8-(2-deoxy-beta-ribofuranosyl)isoxanthopteridine into the human telomeric repeat DNA as a probe for UP1 binding and destabilization of G-tetrad structures.

J.C.Myers, S.A.Moore, Y.Shamoo.

ABSTRACT

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(TTAGGG)n. Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG)2 is approximately 5 nm at 100 mm NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the Tm of the hTR sequence d(TAGGGT)4 from 67.0 degrees C to 36.1 degrees C at physiological conditions (150 mm KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.

Selected figure(s)

Figure 1.
FIG. 1. A, ribbons diagram (50) of UP1 bound to the sequence d(TTAGGGTTAGGG). Two copies of UP1 form a crystallographic dimer in which the DNA is bound in an anti-parallel arrangement across RRM1 (residues 1-92) and RRM2 (residues 93-195) from the two UP1 molecules. The 2-fold rotation of the UP1 molecules into the crystallographic dimer permits the formation of a contiguous single-stranded nucleic acid binding cleft. Previous studies have shown that UP1 has an RNA binding site size of 15 nucleotides, which is in good agreement with the structure. The positions of the 6-MI substitutions in oligonucleotides TR2-6F and TR2-11F are indicated (circles). B, 2F[o] - F[c] electron density from a composite omit map of 6-MI substituted for Gua-11 (TR2-11F) contoured at 1.2 . C, electron density of 6-MI from a 2F[o] - F[c] composite omit map generated using phases obtained from molecular replacement of UP1 bound to d(TTAGGGTTAGGG) contoured at 1.2 . The model shows the position of 6-MI in TR2-6F (blue). The electron density corresponding to the phosphodiester and deoxyribose atoms at position six are of good quality, whereas the highly mobile base appears only weakly. This finding is consistent with electron density maps derived from the wild-type structure that was resolved in our laboratory for comparison. In both the original and substituted DNA, the base at position 11 stacks with Gua-10 and makes interactions through the base O6 (O4 in 6-MI) to the main chain of UP1. In contrast to TR2-6F, the electron density of 6-MI in TR2-11F is moderately well ordered.

Figure 2.
FIG. 2. Fluorescence titration of UP1 with TR2-6F ( o ) and TR2-11F ( . Fluorescent signal of 6-MI is strongly influenced by its environment. In TR2-6F, 6-MI becomes fully unstacked and solvent-exposed upon UP1 binding. In TR2-11F, 6-MI stacks with neighboring Gua-10 when bound to UP1. Excitation and emission wavelengths were 340 and 430 nm, respectively, and both titrations were performed in 20 mM MES, pH 6.0, and 100 mM NaCl at 25 °C. Total gain-of-signal for TR2-6F was 3.3x the base-line; for TR2-11F, it was 1.2x base-line.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 42300-42306) copyright 2003.

Figures were selected by the author.