Structural modifications of RNA influence the 5' exoribonucleolytic hydrolysis by XRN1 and HKE1 of Saccharomyces cerevisiae.
Two 5' exoribonucleases, XRN1 and HKE1, of Saccharomyces cerevisiae have been found to have very important cellular roles, XRN1 playing a key role in mRNA turnover and HKE1 in pre-rRNA processing. Here, an analysis of strong secondary structures in RNA that cause blocks or stalls (accumulation of RNA fragments that are shortened from the 5' end to the site of the secondary structure insertion) in the processive exoribonucleolytic hydrolysis reactions is reported. With both enzymes, oligo(G) tracts of lengths 18, 16, and 9 stall quite effectively, and the stalls are close to the start of the oligo(G) stretch. Two strong stem-loop structures cause measurable but low-level stalls with both enzymes. If the stem-loop structure is placed close to the 5' end of the RNA, substantial inhibition of overall RNA hydrolysis occurs with HKE1 and less, but measurable, inhibition with XRN1. RNA structural modification caused by protein complexing has been investigated by using poly(A) binding protein. The hydrolysis of poly(A) by XRN1 is inhibited by poly(A) binding protein, while HKE1 activity is not affected.
Poole
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Life Sciences Division, Oak Ridge National Laboratory, Tennessee 37831-8080, USA.
Stevens
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D001483:Base Sequence; D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D005095:Exoribonucleases; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C075577:RAT1 protein, S cerevisiae; D003851:Deoxyribonucleases; D005095:Exoribonucleases; C068398:XRN1 protein, S cerevisiae; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I