Activity of 3'-thioAMP derivatives as ribosomal P-site substrates.
The ribosome is a large RNP complex but its main enzymatic activity, the peptidyl transferase, is a ribozyme. As many RNA enzymes use divalent metal ions in catalysis, one of the hypotheses put forward proposed that metal ions might aid peptide bond formation. To be able to test a possible coordination of a metal ion to the 3'-bridging oxygen of P-site substrates, a 3'-thioAMP was synthesized. Its chemical acylation with N-acetyl-L-leucine yielded both mono and diaminoacylated 3'-thioAMP. These thioated substrates were tested for peptide bond formation in an optimized fragment reaction in comparison with their unmodified counterparts. As the amino acid was predominantly linked to the unproductive 2'-OH in AcLeu-thioAMP (5), this substrate was barely active and not used for further analysis. In contrast, Di(AcLeu)-thioAMP (4) was more active than Di(AcLeu)-AMP (2) which is in line with the higher energy of thioesters. Both activities were slightly enhanced when Mn2+ containing buffers were employed in the assay. These data show that thioated P-site substrates are active in peptide bond formation and can in principle be used for metal-ion-rescue experiments in a full translation system.
Dorner
Silke
S
Medizinische Universität Wien, Max F. Perutz Laboratories, University Department at the Vienna Biocenter, Wien, Austria.
Schmid
Walther
W
Barta
Andrea
A
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D007930:Leucine / Q000031:analogs & derivatives* / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008956:Models, Chemical; D014176:Protein Biosynthesis; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism*
D012360:RNA, Transfer, Phe; D013873:Thionucleotides; D000249:Adenosine Monophosphate; D008345:Manganese; D007930:Leucine; C088117:acetylleucine