experimental and theoretical genetic work which established that the code was written in three letter "words" (codons). Today, our understanding of the genetic code permits the prediction of the amino sequence of the protein products of the tens of thousands of genes whose sequences are being determined in genome studies. RNA polymerase is purified The biochemical purification and characterization of RNA polymerase from the bacterium Escherichia coli enabled the understanding of the mechanisms through which RNA polymerase initiates and terminates transcription, and how those processes are regulated to regulate gene expression (i.e. turn genes on and off). Following the isolation of E. coli RNA polymerase, the three RNA polymerases of the eukaryotic nucleus were identified, as well as those associated with viruses and organelles. Studies of transcription also led to the identification of many protein factors that influence transcription, including repressors, activators and enhancers. The availability of purified preparations of RNA polymerase permitted investigators to develop a wide range of novel methods for studying RNA in the test tube, and led directly to many of the subsequent key discoveries in RNA biology. 1966–1975 First complete nucleotide sequence of a biological nucleic acid molecule Although determining the sequence of proteins was becoming somewhat routine, methods for sequencing of nucleic acids were not available until the mid-1960s. In this seminal work, a specific tRNA was purified in substantial quantities, and then sliced into overlapping fragments using a variety of ribonucleases. Analysis of the detailed nucleotide composition of each fragment provided the information necessary to deduce the sequence of the tRNA. Today, the sequence analysis of much larger nucleic acid molecules is highly automated and enormously faster. Evolutionary variation of homologous RNA sequences reveals folding patterns Additional tRNA molecules were purified and sequenced. The first comparative sequence analysis was done and revealed that the sequences varied through evolution in such a way that all of the tRNAs could fold into very similar secondary structures (two-dimensional structures) and had identical sequences at numerous positions (e.g. CCA at the 3′ end). The radial four-arm structure of tRNA molecules is termed the 'cloverleaf structure', and results from the evolution of sequences with common ancestry and common biological function. Since the discovery of the tRNA cloverleaf, comparative analysis of numerous other homologous RNA molecules has led to the identification of common sequences and folding patterns. First complete genomic nucleotide sequence The 3569 nucleotide sequence of all of