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Contributions of the individual domains in human La protein to its RNA 3'-end binding activity.

Title data

Ohndorf, Uta-Maria ; Steegborn, Clemens ; Knijff, Rainer ; Sondermann, Peter:
Contributions of the individual domains in human La protein to its RNA 3'-end binding activity.
In: The Journal of Biological Chemistry. Vol. 276 (20 July 2001) Issue 29 . - pp. 27188-27196.
ISSN 1083-351X
DOI: https://doi.org/10.1074/jbc.M102891200

Abstract in another language

The autoantigen La regulates the maturation of RNA polymerase III transcripts by binding to their poly(U) termination signal. The modular protein harbors a N-terminal RNA recognition motif (RRM), RRM1, and in the C-terminal domain, a second, atypical RRM2, in addition to a phosphorylation site, and a putative nucleotide binding site. This study presents a detailed investigation into the RNA 3'-end binding properties of La by using binding titration and competition assays with subsequent gel mobility shift analysis. Two truncation mutants containing one (La-RRM1) or both (La-RRM1-RRM2) RNA-binding domains were constructed, overexpressed, and purified. A K(d) value of 25 +/- 10 nm for La binding to a nonameric RNA ligand with the oligouridylate recognition sequence was obtained, discriminating with a specificity ratio of approximately 100 for this probe over a RNA ligand with a 3'-poly(A) stretch. The N-terminal La-RRM1 region was identified as the major contributor of these properties to La, manifested in a 5-fold lower K(d) of 5 +/- 3 nm and a slightly increased specificity ratio of 120 for the RNA ligand. The atypical RRM2 in the C-terminal domain of La has an unprecedented negative effect on 3'-end RNA recognition, as indicated by a higher K(d) value of 90 +/- 10 nm for the La-RRM1-RRM2 mutant but comparable specificity. Thus the C-terminal regions beyond RRM2 positively modulate the RNA binding affinity of La. Negative regulation, however, occurs through Ser(366) phosphorylation decreasing the binding affinity by 2-fold. ATP had no influence on RNA complex formation. The functional implications of these findings for the mechanism of action of La are discussed.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 11342556
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry > Chair Biochemistry - Univ.-Prof. Dr. Clemens Steegborn
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 13 Apr 2015 10:16
Last Modified: 13 Apr 2015 10:16
URI: https://eref.uni-bayreuth.de/id/eprint/10098