The ribosome is the catalyst of peptide bond formation within all living organisms. The 50S ribosomal subunit of the ribosome contains the peptidyl transferase center where the peptide bonds are formed. The recent high resolution crystal structure of the intact 50S subunit suggests that the peptidyl transferase center is composed exclusively of rRNA. The unusual chemical basicity of a single nucleotide within the active site may allow it to serve as a general acid-base catalyst for peptide bond formation. This implies that the ribosome is an RNA enzyme or ribozyme, though this has yet to be biochemically demonstrated. The specific objective of this project is to demonstrate peptidyl transferase activity by an in vitro transcribed 23S rRNA in the absence of ribosomal proteins. To achieve this objective a new version of the peptidyl transferase reaction that alleviates the artifactual reaction conditions required of the previously used fragment reaction will be utilized. The structure of the 50S subunit will be employed to design truncated 23S rRNA fragments that are likely to fold into an active conformation. These experiments are capable of providing definitive biochemical evidence to establish whether 23S rRNA is the catalyst of protein synthesis and will provide a biochemically amenable system for the analysis of the ribosomal reaction mechanism.