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Effect of pre‐tRNA 5’ leader sequence variation on the thermodynamic coupling and shared molecular recognition between RNA and protein components of RNase P
Author(s) -
Niland Courtney Nicole,
Yandek Lindsay E.,
Guenther UlfPeter,
Jankowsky Eckhard,
Harris Michael E.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.777.2
Subject(s) - ribozyme , rnase p , rna , hammerhead ribozyme , ribonucleoprotein , rnase h , biology , transfer rna , nucleotide , signal recognition particle rna , biochemistry , rna binding protein , chemistry , gene
Ribonuclease P, RNase P, recognizes different precursor tRNAs to process the 5’ ends of each and produce mature tRNAs. The RNA subunit of this ribonucleoprotein enzyme is catalytic, however, the protein subunit is essential in vivo and contacts nucleotides in the 5’ leader. Extensive genomic variation of this sequence makes identification of cognate binding motifs difficult. Since the RNA subunit also recognizes this region, we focus on the shared molecular recognition of the 5’ leader by RNA and protein. We developed a new method that allows us to quantify rate constants of individual substrates in a reaction with many alternative substrates. This allows analysis of all possible sequences in areas of protein and RNA contact, providing information on thermodynamic coupling between nucleotides. The effect of randomizing the protein binding site or both RNA and protein binding sites was determined. The results confirm that randomizing the protein binding site alters the of the holoenzyme reaction but not the ribozyme. Randomizing nucleotides at both protein and RNA binding sites alters the kinetics of both the ribozyme and holoenzyme. Illumina sequencing of the residual substrates during the reaction allows us to calculate the rate constants for each sequence. This data will reveal whether nucleotides at RNA‐RNA binding sites influence the thermodynamic contribution of those at protein‐RNA contacts and vice versa.