Characterization of the pre-mRNA binding site for yeast ribosomal protein L32: the importance of a purine-rich internal loop.
The structure of the RNA binding target for Saccharomyces cerevisiae ribosomal 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.
Li
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Department of Chemistry, Bryn Mawr College, PA 19010, USA.
Dalal
S
S
Kohler
J
J
Vilardell
J
J
White
S A
SA
D000814:Aniline Compounds / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D017608:Chromium Compounds / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005038:Ethylnitrosourea / Q000494:pharmacology; D015951:Factor Xa / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010713:Phosphatidylcholines / Q000494:pharmacology; D011485:Protein Binding; D012322:RNA Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D017423:Sequence Analysis, RNA; D013696:Temperature; D013816:Thermodynamics
D000814:Aniline Compounds; D017608:Chromium Compounds; D005296:Ferrous Compounds; D010713:Phosphatidylcholines; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C041806:ribosomal protein L32; C055526:Fe(II)-EDTA; D004492:Edetic Acid; D012260:Ribonucleases; D015951:Factor Xa; C017251:1,2-oleoylphosphatidylcholine; D005038:Ethylnitrosourea; C023650:aniline