GMP synthetase (EC 6.3.4.1), a glutamine amido-transferase encoded by the guaA gene, catalyzes the synthesis of GMP from XMP. The guaA gene was subcloned from the Clarke and Carbon (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) plasmid pLC34-10, and the nucleotide sequence was determined. The structural gene encodes a protein of 525 amino acid residues having a calculated Mr of 58,604. The amino acid sequence of the NH2 terminus of GMP synthetase was determined and used to verify the translation start site determined from the DNA sequence. A 68-base pair intercistronic region separates guaA from the upstream guaB gene in the polycistronic guaBA operon. The 3' end of the guaA mRNA was determined by S1 nuclease mapping. The 3' end of guaA mRNA is 36-37 nucleotides downstream of the translation stop codon within a region of dyad symmetry that resembles a rho-independent transcription termination site.