To investigate the relationship between tRNA conformation and specific recognition by aminoacyl-tRNA synthetases, a full-length tRNA molecule was assembled by annealing together two oligonucleotides representing fragments of Escherichia coli tRNA(Pro). A shorter chemically synthesized 5'-fragment (7-18 nucleotides) was combined with an in vitro transcribed 3'-fragment (59 nucleotides). Despite a break in the phosphodiester backbone between nucleotides U17a and G18, this tRNA molecule was an efficient substrate for class II Escherichia coli proline tRNA synthetase. While the deletion of three D-loop nucleotides (U17a, U17, and C16) was tolerated, removal of G15 and A14 significantly reduced aminoacylation efficiency. Hybrid DNA-RNA "annealed" substrates were also prepared and assayed for aminoacylation. Native gel electrophoresis was used to compare the global folding of the various substrates tested. The results of these studies suggest that proline tRNA synthetase is sensitive to changes in the core region of tRNA(Pro) through which information required for efficient aminoacylation may be transmitted. In particular, nucleotides in the D-loop and backbone functional groups in the D-stem appear to be critical for maintaining a tRNA structure that is optimal for recognition by proline tRNA synthetase in vitro.