Structure of PTB RRM1(41-163) bound to an RNA stemloop containing a structured loop derived from viral internal ribosomal entry site RNA

Complex of PTB RRM1 bound to stemloop RNA containing UCUUU loop sequence.

Chemical shifts for RNA imino protons were obtained from NOESY spectra measured at 5C.

3D f1-13Cfiltered,f2-13C-edited NOESY aliphatic-HSQC

separate tube (no insert) not similar to the experimental sample tube

C-terminal helix folding upon pyrimidine-rich hairpin binding to PTB RRM1. Implications for PTB function in Encephalomyocarditis virus IRES activity.

RNA (5'-R(*GP*GP*GP*AP*CP*CP*UP*GP*GP*UP*CP*UP*UP*UP*CP*CP*AP*GP*GP*UP*CP*CP*C)-3')

PTB RRM1 corresponding to residues 41-163 was subcloned into pTYB11 which is part of the IMPACT-CN system. A C-terminal intein tag was included in the construct which self cleaves on a chitin column. E. coli strain BL21 (DE3) codonPlus-RIL was transformed and overexpression using M9 media containing 13C6-glucose and 15NH4Cl produced isotope-labeled protein.

in vitro transcription from chemically synthesized nucleotides with a Pro266Leu mutant version of RNA T7 polymerase. Both unlabeled and 13C,15N-labeled NTPs were produced in-house. RNA were purified by anionic exchange in denaturing conditions.

PTB RRM1 forms an additional C-terminal helix upon binding to an RNA hairpin containing a pyrimidine-rich structured loop.

Standard 3D NOESYs measured with 150 msec mixing time. 2D NOESY with 200 msec mixing time.
NOESYs measured at 5C were obtained using 3919-watergate sequence before detection.
RNA Constraints for all base-paired nucleotides except the closing G9-C15 basepair of the
loop were derived from NOESY spectra obtained with free RNA - the NOE patterns were the
same as for the RNA in the complex but signal to noise was better. 3D NOESYs of unlabeled
RNA were measured with 200 msec mixing time, and 2D NOESY of unlabeled RNA was measured
with 250 msec mixing time.

simulated annealing, simulated annealing

The data for N-H imino signals of the SL23H RNA were obtained from TROSY experiments
measured at 5C where these signals were visible.

NMR structure of PTB RRM1 (54-147) in the free state.
NMR structure of PTB RRM1(49-146) bound to single-stranded CUCUCU RNA
Pf1 phage added for orienting macromolecules to obtain RCS. Compared to sample without Pf1 phage.
ribose at positions 9,11,13 and 15 are 13C,15N-labeled.
Only cytosine and uracil nucleotides are uniformly 13C,15N-labeled.
[U-13C; U-15N]-ribose-Cyt9,Ura11,Ura13,Gua15

UNIO is an all in one suite with utility programs for automated resonance
assignment, peak-picking, NOE assignment, and constraint collection.
It can be used together with structure calculation programs for completely
automated structure determination from NMR spectra.

Case, Darden, Cheatham, III, Simmerling, Wang, Duke, Luo, ... and Kollman