5′‐Terminal Nucleotide Sequence of the Messenger RNA Coding for Bovine Corticotropin/β‐Lipotropin Precursor

Abstract

The complete 5'-terminal nucleotide sequence of the MRNA coding for the bovine common precursor of corticotropin and beta-lipotropin has been determined. The 5'-32P-labelled, 21-nucleotides-long, single-stranded DNA fragment complementary to a portion of the 5'-noncoding region of the mRNA was prepared from a cDNA clone and elongated by reverse transcriptase reaction with the mRNA as template. The DNA transcript formed was sequenced by the procedure of Maxam and Gilbert, and the resultant sequence was cross-checked by two-dimensional electrophoretic analysis of the partial alkaline digest of the 5'-32P-labelled mRNA. The 5'-terminal nucleotide residue was determined by two-dimensional thin-layer chromatography of the complete hydrolysis product of the 5'-32P-labelled mRNA. The nucleotide sequence determined, which partially overlaps the known sequence of the cloned cDNA, reveals the complete 5'-terminal sequence of the mRNA. This, in conjunction with our previous data, defines the complete primary structure of the mRNA. The mRNA is composed of 1098 nucleotides, including an unusually long 5'-noncoding sequence of 128 nucleotides. The presence of a 'cap' structure at the 5' terminus of the mRNA is suggested. The 5'-terminal 48 nucleotide residues of the mRNA are extremely purine-rich, having an A + G content of 83%, whereas all pyrimidine-rich segments are located downstream from there. Because the 5'-noncoding region of the mRNA contains three segments of potential secondary structure which partially overlap, it can exist in a number of alternative base-pairing configurations. However, its interaction with the 3'-terminal segment of 18-S rRNA at the site of maximal complementarity would fix the mRNA configuration in such a way as to bring the possible site of ribosome binding near the initiation codon.