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The 3′ end CCA of mature tRNA is an antideterminant for eukaryotic 3′-tRNase

Published online by Cambridge University Press:  01 February 1999

AMULYA MOHAN ,
STACEY WHYTE ,
XUDONG WANG ,
MASAYUKI NASHIMOTO  and
LOUIS LEVINGER
AMULYA MOHAN
Affiliation:
Department of Natural Sciences/Biology, York College, City University of New York, 94-20 Guy R. Brewer Boulevard, Jamaica, New York 11451, USA
STACEY WHYTE
Affiliation:
Department of Natural Sciences/Biology, York College, City University of New York, 94-20 Guy R. Brewer Boulevard, Jamaica, New York 11451, USA Present address: New York College of Osteopathic Medicine, Old Westbury, New York 11568, USA.
XUDONG WANG
Affiliation:
Department of Natural Sciences/Biology, York College, City University of New York, 94-20 Guy R. Brewer Boulevard, Jamaica, New York 11451, USA
MASAYUKI NASHIMOTO
Affiliation:
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700, USA
LOUIS LEVINGER
Affiliation:
Department of Natural Sciences/Biology, York College, City University of New York, 94-20 Guy R. Brewer Boulevard, Jamaica, New York 11451, USA Biology Program/CUNY Graduate School
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Abstract

Cytoplasmic tRNAs undergo posttranscriptional 5′ and 3′ end processing in the eukaryotic nucleus, and CCA (which forms the mature 3′ end of all tRNAs) must be added by tRNA nucleotidyl transferase before tRNA can be aminoacylated and utilized in translation. Eukaryotic 3′-tRNase can endonucleolytically remove a 3′ end trailer by cleaving on the 3′ side of the discriminator base (the unpaired nucleotide 3′ of the last base pair of the acceptor stem). This reaction proceeds despite a wide range in length and sequence of the 3′ end trailer, except that mature tRNA containing the 3′ terminal CCA is not a substrate for mouse 3′-tRNase (Nashimoto, 1997, Nucleic Acids Res 25:1148–1154). Herein, we extend this result with Drosophila and pig 3′-tRNase, using Drosophila melanogaster tRNAHis as substrate. Mature tRNA is thus prevented from recycling through 3′ end processing.

We also tested a series of tRNAs ending at the discriminator base (−), with one C added (+C), two Cs added (+CC), and CCA added (+CCA) as 3′-tRNase inhibitors. Inhibition was competitive with both Drosophila and pig 3′-tRNase. The product of the 3′-tRNase reaction (−) is a good 3′-tRNase inhibitor, with a KI approximately two times KM for the normal 3′-tRNase substrate. KI increases with each nucleotide added beyond the discriminator base, until when tRNA+CCA is used as inhibitor, KI is approximately forty times the substrate KM. The 3′-tRNase can thus remain free to process precursors with 3′ end trailers because it is barely inhibited by tRNA+CCA, ensuring that tRNA can progress to aminoacylation. The active site of 3′-tRNase may have evolved to make an especially poor fit with tRNA+CCA.

Keywords

eukaryotic 3′-tRNasemature tRNAtRNA end-processing kinetics
Type
Research Article
Information
RNA , Volume 5 , Issue 2 , February 1999 , pp. 245 - 256
Copyright
1999 RNA Society

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