Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Abstract

Pseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec À1 . In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (k C ) by TruB was determined to be 0.5 sec À1 . This rate constant is similar to the k cat obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec À1 , respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism.

RNA modification enzyme TruB is a tRNA chaperone

“…S1); thus, TruB assists in tRNA folding. Next, the experiment was repeated with the TruB D48N variant, which is inactive in pseudouridine formation but unimpaired in tRNA binding (18). The catalytically inactive TruB variant is able to fold tRNA at the same rate as TruB WT, demonstrating that TruB's tRNA chaperone activity is independent of its tRNA modification activity.…”

“…Single-turnover pseudouridylation experiments (Fig. S3C) revealed that the TruB K64E variant is most affected: it is 750-fold slower than the WT enzyme (Table S1) (17)(18)(19). Therefore, all further experiments were conducted with the TruB K64E variant.…”

“…† Determined in ref. 18. Phe assessed by CMCT modification in E. coli WT and truB KO strains expressing TruB variants.…”

“…Superimposing the T-arm bound to TruB onto full-length tRNA indicates that tRNA binding by TruB disrupts the tertiary interactions between the T-and D-arm of tRNA. Kinetic studies in our laboratory have shown that tRNA binds relatively quickly to TruB but that catalysis of pseudouridine formation is surprisingly slow and rate limiting (18). In fact, slow catalysis is a hallmark of all pseudouridine synthases studied in detail thus far (18)(19)(20).…”

“…Kinetic studies in our laboratory have shown that tRNA binds relatively quickly to TruB but that catalysis of pseudouridine formation is surprisingly slow and rate limiting (18). In fact, slow catalysis is a hallmark of all pseudouridine synthases studied in detail thus far (18)(19)(20). Deleting the E. coli truB gene does not impact growth under optimal conditions (10).…”