Toward ribosomal RNA catalytic activity in the absence of protein.
The ribosome is the ribonucleoprotein particle responsible for translation of genetic information into proteins. The RNA component of the ribosome has been implicated as the catalytic entity for peptide bond formation based on protease resistance and structural data indicating an all-RNA active site. Nevertheless, peptidyl transfer by ribosomal RNA (rRNA) alone has not been demonstrated. In an attempt to show such activity we generated a minimal construct that comprises much of the 23S rRNA peptidyl transferase center, including the central loop and the A- and P-loops. This minimal rRNA domain was inactive in peptide bond formation under all conditions tested. The RNA was subsequently subjected to six rounds of in vitro selection designed to enrich for this activity. The result was a mutated rRNA sequence that could catalyze the covalent linkage of an A-site and P-site substrate; however, the product did not contain a peptide bond. The current study is an example of an in vitro derived alternate function of rRNA mutants and illustrates the evolutionary possibility that the protoribosome may have used amino acids as substrates before it gained the ability to join them into peptides. Though peptidyl transferase activity in the absence of protein remains elusive, the ease with which alternate catalytic activity was selected from rRNA with a small number of mutations suggests that rRNA may have inherent activity. This study represents a step on the path toward isolating that native activity.
Anderson
Rachel M
RM
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
Kwon
Miyun
M
Strobel
Scott A
SA
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D001411:Geobacillus stearothermophilus / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052156:SELEX Aptamer Technique; D017423:Sequence Analysis, RNA
D001426:Bacterial Proteins; D016337:RNA, Catalytic; D012335:RNA, Ribosomal