Abstract
tRNAs are transcribed as precursors and processed in a series of reactions culminating in aminoacylation and translation. Central to tRNA maturation, the 3' end trailer can be endonucleolytically removed by tRNase Z. A flexible arm (FA) extruded from the body of tRNase Z consists of a structured alphaalphabetabeta hand that binds the elbow of pre-tRNA. Deleting the FA hand causes an almost 100-fold increase in Km with little change in kcat, establishing its contribution to substrate recognition/binding. Remarkably, a 40-residue Ala scan through the FA hand reveals a conserved leucine at the ascending stalk/hand boundary that causes practically the same increase in Km as the hand deletion, thus nearly eliminating its ability to bind substrate. Km also increases with substitutions in the GP (alpha4-alpha5) loop and at other conserved residues in the FA hand predicted to contact substrate based on the co-crystal structure. Substitutions that reduce kcat are clustered in the beta10-beta11 loop.
Highlights
The 3Ј end CCA is an anti-determinant for tRNase Z that discourages the recycling of mature tRNAs (4 –7), not in every case (8)
The co-crystal structure shows contacts principally between ␣5 residues in the hand of the flexible arm (FA) and the D/T loops of tRNA (20). These results demonstrate that the FA of tRNase Z is involved with substrate recognition and binding remote from its active site and from the scissile bond of the substrate
Results with tRNase Z ⌬FA establish that the hand of the FA contributes almost two orders of magnitude to the base Km for wild type tRNase Z of 3.4 ϫ 10Ϫ8 M
Summary
TRNase Z Mutagenesis and Expression—D. melanogaster tRNase Z cDNA (accession number AY119279) was baculovirus-expressed from methionine 24 (suggested to be the translation start for the nuclear form of the enzyme) (32). Variant enzymes were used in two or more kinetic experi- To further probe for residues and regions important for subments (typically n ϭ 3– 4); a parallel experiment with wild type strate recognition and binding, a FA hand deletion and an Ala tRNase Z was included each time variant processing kinetics scan through the hand of D. melanogaster tRNase Z Z performed on the same day and, do not coincide lines in Fig. 1) was singly substituted with alanine, expressed with values calculated using the wild type tRNase Z data pre- using baculovirus, tested for processing efficiency (data not sented on row 1 of supplemental Table 1 (the means of all eighty shown), and compared with the wild type enzyme in repeated. Because Km is roughly equivalent to an enzyme-substrate dissociation constant (KD), the hand of the FA contributes up to two orders of magnitude to the recognition/binding of substrate by wild type tRNase Z, which displays a Km of ϳ3.7ϫ 10Ϫ8 M (supplemental Table 1)
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