https://www.cell.com/cell/pdf/0092-8674(78)90039-9.pdf

How do eucaryotic ribosomes select initiation regions in messenger RNA

T4 RNA LIGASE catalyses the formation of an internucleotide phosphodiester bond between an oligonucleotide donor molecule with a 5′-terminal phosphate and an oligonucleotide acceptor molecule with a 3′-terminal hydroxyl1–3. Although the minimal acceptor must be a trinucleoside diphosphate, dinucleoside pyrophosphates and mononucleoside 3′,5′-bisphosphates (pNps) are effective donors in the intermolecular reaction4–6. We demonstrate here that various high molecular weight RNA molecules are acceptors in the RNA ligase reaction even when present in very low concentrations in the reaction mixture. One immediate consequence of this observation is that a convenient method for labelling the 3′ end of RNA molecules in vitro becomes available. By using a [5′-32P]pNp as a donor and RNA as an acceptor, the product of the reaction is an RNA molecule one nucleotide longer, with a 3′-terminal phosphate and a 32P-phosphate in the last internucleotide linkage. This reaction is therefore analogous to the in vitro labelling of the 5′ termini of RNA chains with polynucleotide kinase and [γ-32P]ATP and can be used in situations where 5′ labelling is not possible. In addition, the ability to add various donors to an RNA molecule should allow the function of the 3′ terminus of the molecule to be investigated.

3'-terminal labelling of RNA with T4 RNA ligase.

The viroid of the potato spindle tuber disease (PSTV) is a covalently closed ring of 359 ribonucleotides. As a result of intramolecular base pairing, a serial arrangement of double-helical sections and internal loops form a unique rod-like secondary structure. PSTV is the first pathogen of a eukaryotic organism for which the complete molecular structure has been established.

Nucleotide sequence and secondary structure of potato spindle tuber viroid

Complete Nucleotide Sequence of the Japanese Encephalitis Virus Genome RNA

Publisher Summary This chapter describes a general method, which has made possible the sequence analysis of tRNAs from mitochondria, chloroplasts, and a wide variety of prokaryotic and eukaryotic organisms using in some cases as little as 20μg of purified tRNA. The development of in vitro 32 P-labeling techniques has now permitted sequence analysis of nonradioactive tRNAs utilizing adaptations of methods developed by Sanger and co-workers for analysis of uniformly 32 P-labeled RNAs. oligonucleotides with a free 5'-hydroxyl end which are present in digests of nonradioactive nucleic acids can be labeled to high specific activity using T4 polynucleotide kinase and [γ- 32 P]ATP. This approach is used for labeling with 32 P the 5' ends of both mononucleotides present in complete tRNA hydrolysates and oligonucleotides present in T1 or pancreatic RNase digests of tRNA. Using oligonucleotides present in tRNAs of known sequence as standards, the mobility shifts of many commonly occurring modified nucleotides are characterized, which has proved useful in recognizing these modified nucleotides within unknown sequences. The basic steps in the sequence analysis of a nonradioactive tRNA are summarized in the chapter.

A.M. Gillum

Papers

Nucleotide sequence of salmon testes and salmon liver cytoplasmic initiator tRNA.

Nucleotide sequence of human placenta cytoplasmic initiator tRNA