Hfq stimulates the activity of the CCA-adding enzyme

Abstract

Background: The bacterial Sm-like protein Hfq is known as an important regulator involved in many reactions of RNA metabolism. A prominent function of Hfq is the stimulation of RNA polyadenylation catalyzed by E. coli poly(A) polymerase I (PAP). As a member of the nucleotidyltransferase superfamily, this enzyme shares a high sequence similarity with an other representative of this family, the tRNA nucleotidyltransferase that synthesizes the 3'-terminal sequence C-C-A to all tRNAs (CCA-adding enzyme). Therefore, it was assumed that Hfq might not only influence the poly(A) polymerase in its specific activity, but also other, similar enzymes like the CCA-adding enzyme.

Results: Based on the close evolutionary relation of these two nucleotidyltransferases, it was tested whether Hfq is a specific modulator acting exclusively on PAP or whether it also influences the activity of the CCA-adding enzyme. The obtained data indicate that the reaction catalyzed by this enzyme is substantially accelerated in the presence of Hfq. Furthermore, Hfq binds specifically to tRNA transcripts, which seems to be the prerequisite for the observed effect on CCA-addition.

Conclusion: The increase of the CCA-addition in the presence of Hfq suggests that this protein acts as a stimulating factor not only for PAP, but also for the CCA-adding enzyme. In both cases, Hfq interacts with RNA substrates, while a direct binding to the corresponding enzymes was not demonstrated up to now (although experimental data indicate a possible interaction of PAP and Hfq). So far, the basic principle of these stimulatory effects is not clear yet. In case of the CCA-adding enzyme, however, the presented data indicate that the complex between Hfq and tRNA substrate might enhance the product release from the enzyme.

Figure 1

CCA-addition is stimulated by Hfq. (A) The E. coli CCA-adding enzyme was incubated for indicated times with radioactively labeled yeast tRNA^Phe without CCA-end as a substrate in the absence or presence of Hfq or BSA, respectively. The reaction products were separated by denaturing polyacrylamide gel electrophoresis. CCA-addition leads to a reduced electrophoretic mobility of the labeled tRNA, and the corresponding signal intensities indicate a dramatic enhancement of the CCA incorporation in the presence of Hfq, while the CCA synthesis without Hfq or BSA addition was only moderate. BSA also led to a considerable stimulation, probably by stabilizing the active CCA-adding enzyme. These results were verified using different tRNA substrates (E. coli tRNA^Ala, phage T5 tRNA^Cys, not shown). M, mock incubation without addition of CCA-adding enzyme; -, activity of CCA-adding enzyme without any additional protein. (B) CCA-addition in the presence of several RNA binding proteins, BSA, Hfq or Hfq variants. Only Hfq and the two variants V43R and K56A lead to a strong increase in CCA-addition, while all other RNA binding proteins show a much weaker stimulating effect, indistinguishable to that of BSA. NusA: transcription elongation factor (E. coli); TGT: tRNA guanine transglycosylase (Z. mobilis); HU: histone-like protein that also interacts with RNA (E. coli); P: RNase P protein subunit (E. coli).

Figure 2

Gel shift experiments with CCA-adding enzyme, Hfq and BSA. (A) Only Hfq (either alone or in the presence of CCA-adding enzyme) was binding to the radioactively labeled tRNA substrate (without CCA terminus), leading to a reduced electrophoretic mobility on a native polyacrylamide gel (arrow). The identical band shifts in the gel after sample preincubation for 1 and 10 minutes indicate that the binding equilibrium was reached within 1 minute and that the Hfq-tRNA interaction is rather stable over time. (B) The tRNA/Hfq complex was competed by a nonspecific plasmid run-off transcript (left) or a transcript corresponding to the 3'-end of rpsO mRNA. While the rpsO RNA (carrying an Hfq binding site) efficiently replaced the bound tRNA at concentrations above 100 fmol (> 5-fold excess), the plasmid transcript could not compete for binding at any concentration, indicating a specific interaction of Hfq with the tRNA.

Figure 3

Apparent kinetic parameters of CCA-addition. Increasing amounts of unlabeled tRNA^Phe were incubated with CCA-adding enzyme, NTPs and α-³²P-ATP in presence (CCA+Hfq (blue diamonds), CCA+BSA (black triangles)) or absence (CCA, red bullets) of Hfq or BSA. Reaction products were separated by PAGE and analyzed by autoradiography. The corresponding reaction velocities were determined using GraphPadPrism software.