Characterization of the Pre-mRNA Binding Site for Yeast Ribosomal Protein L32: The Importance of a Purine-rich Internal Loop

Hu Li, Seema Dalal, Jennifer Kohler, Josep Vilardell, Susan A. White

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The structure of the RNA binding target forSaccharomyces cerevisiaeribosomal protein L32 was examined using chemical and enzymatic probes as well as thermodynamic methods.In vivo, the production of yeast RPL32 is regulated by a feedback mechanism whereby RPL32 binds to the 5′ end of its transcript and inhibits splicing. The binding site of ribosomal protein L32 on the L32 RNA transcript can be reduced to fewer than 30 nucleotides which compromise a stem-internal loop-stem structural motif. The internal loop is closed by a potential G·U pair, is asymmetric and contains mostly purines. The existence of the two helical regions was confirmed by chemical and enzymatic probing. The reactivity of the loop region suggests a structure intermediate between that of single and double-stranded RNA. Base stacking continues into the loop, but two loop bases are extremely reactive to chemical agents. The interaction between the model RNA and the protein is specific and has a dissociation constant of approximately 10 nM. Several of the loop bases are critical for protein binding, as demonstrated by mutational data and chemical protection and modification interference studies. The internal loop destabilizes the RNA, and allows the RNA to melt in an all-or-none fashion.

Original languageEnglish (US)
Pages (from-to)447-459
Number of pages13
JournalJournal of Molecular Biology
Volume250
Issue number4
DOIs
StatePublished - Jul 21 1995

Fingerprint

Fungal Proteins
RNA Precursors
Binding Sites
RNA
Computer Security
Purines
Double-Stranded RNA
Thermodynamics
Protein Binding
Proteins
Nucleotides
Yeasts
purine
ribosomal protein L32

Keywords

  • internal loops
  • purine mismatches
  • RNA protein binding
  • RNA secondary structure
  • RNA stability

ASJC Scopus subject areas

  • Molecular Biology
  • Virology

Cite this

Characterization of the Pre-mRNA Binding Site for Yeast Ribosomal Protein L32 : The Importance of a Purine-rich Internal Loop. / Li, Hu; Dalal, Seema; Kohler, Jennifer; Vilardell, Josep; White, Susan A.

In: Journal of Molecular Biology, Vol. 250, No. 4, 21.07.1995, p. 447-459.

Research output: Contribution to journalArticle

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