3'-end labeling of RNA with recombinant yeast poly(A) polymerase.
Two commonly used methods to end-label RNA-molecules are 5'-end labeling by polynucleotide kinase and 3'-end labeling with pCp and T4 RNA ligase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepin 5'-triphosphate (3'-deoxy-ATP) which is added by poly(A) polymerase. For a synthetic RNA it is shown that 40% of cordycepin becomes incorporated when the nucleotide is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at its 3'-end. The reaction is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNA-molecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase.
Lingner
J
J
Department of Cell Biology, Biozentrum of the University of Basel, Switzerland.
Keller
W
W
D001483:Base Sequence; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism*; D011130:RNA Ligase (ATP) / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D003838:Deoxyadenine Nucleotides; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; C011746:3'-deoxyadenosine 5'-triphosphate; D012313:RNA; D011062:Polynucleotide Adenylyltransferase; D011130:RNA Ligase (ATP)