Regulation of substrate recognition by the MiaA tRNA prenyltransferase modification enzyme of Escherichia coli K-12

We purified polyhistidine (His(6))-tagged and native Escherichia coli MiaA tRNA prenyltransferase, which uses dimethylallyl diphosphate (DMAPP) to isopentenylate A residues adjacent to the anticodons of most tRNA species that read codons starting with U residues. Kinetic and binding studies of purified MiaA were performed with several substrates, including synthetic wild-type tRNA(Phe), the anticodon stem-loop (ACSL(Phe)) of tRNA(Phe), and bulk tRNA isolated from a miaA mutant. Gel filtration shift and steady-state kinetic determinations showed that affinity-purified MiaA had the same properties as native MiaA and was completely active for tRNA(Phe) binding. MiaA had a K-m(app) (tRNA substrates) approximate to 3 nM, which is orders of magnitude lower than that of other purified tRNA modification enzymes, a K-m(app) (DMAPP) = 632 nM, and a k(cat)(app) = 0.44 s(-1). MiaA activity was minimally affected by other modifications or nonsubstrate tRNA. species present in bulk tRNA isolated from a miaA mutant. MiaA modified ACSL(Phe) with a k(cat)(app)/K-m(app) substrate specificity about 17-fold lower than that for intact tRNA(Phe) mostly due to a decrease in apparent substrate binding affinity. Quantitative Western immunoblotting showed that MiaA is an abundant protein in exponentially growing bacteria (660 monomers per cell; 1.0 mu M concentration) and is present in a catalytic excess. However, MiaA activity was strongly competitively inhibited for DMAPP by ATP and ADP (K-i(app) = 0.06 mu M), suggesting that MiaA activity is inhibited substantially in vivo and that DMAPP may bind to a conserved P-loop motif in this class of prenyltransferases. Band shift, filter binding, and gel filtration shift experiments support a model in which MiaA tRNA substrates are recognized by binding tightly to MiaA multimers possibly in a positively cooperative way (K-d(app) approximate to 0.07 mu M).