ABSTRACT
The dynamic regulation of biological processes by RNA has emerged as a key field in recent years, and was the topic of the 62nd Mosbacher Colloquium of the German Society for Biochemistry and Molecular Biology (GBM). The 2011 Colloquium, held in April in the romantic Neckar-river region, was also a celebration of the tenth anniversary of the RNA Biochemistry study group within the GBM, which acts as platform for RNA biologists and chemists within Germany and in other European countries.
Subject(s)
RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA/genetics , RNA/metabolism , Biochemistry/methods , Gene Expression Profiling/methods , Gene Expression Regulation , Humans , Molecular Biology/methods , RNA/chemistryABSTRACT
tRNA-nucleotidyltransferases are fascinating and unusual RNA polymerases responsible for the synthesis of the nucleotide triplet CCA at the 3'-terminus of tRNAs. As this CCA end represents an essential functional element for aminoacylation and translation, these polymerases (CCA-adding enzymes) are of vital importance in all organisms. With a possible origin of ancient telomerase-like activity, the CCA-adding enzymes obviously emerged twice during evolution, leading to structurally different, but functionally identical enzymes. The evolution as well as the unique polymerization features of these interesting proteins will be discussed in this review.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , RNA Nucleotidyltransferases/metabolism , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/classification , Evolution, Molecular , Protein Conformation , RNA Nucleotidyltransferases/chemistry , RNA Nucleotidyltransferases/classification , Substrate SpecificityABSTRACT
The 5'-triphosphates of 2'-hydroxymethyluridine (2'-homouridine) and 2'-hydroxyethyluridine were prepared from the corresponding acetyl-protected nucleosides by initial phosphitylation with 2-chloro-5,6-benzo-1,2,3-dioxaphosphorin-4-one. 2'-Acetamidouridine 5'-triphosphate was prepared in an analogous fashion from uridine 2'-C-, 3'-O-gamma-butyrolactone, in which the 3'-hydroxyl group is internally protected as the lactone. Subsequent treatment with ammonia gave the required acetamido triphosphate. All three triphosphates were investigated as substrates for T7 RNA polymerase and a Y639F mutant of this enzyme. 2'-Homouridine triphosphate was found to be a substrate for the wild-type enzyme in the presence of manganese and was specifically incorporated into short RNA transcripts (20 and 21 nucleotides in length). The presence of the analogue within the transcripts was confirmed through its resistance to alkaline hydrolysis. Gel electrophoretic analysis also showed that 2'-homouridine could be multiply incorporated into a transcript with a length of 75 nucleotides. This is the first report of a 2'-deoxy-2'-alpha-C-branched nucleoside 5'-triphosphate acting as a substrate for T7 RNA polymerase. The 2'-hydroxyethyl- and 2'-acetamido -uridine triphosphates were not substrates for the enzymes.
