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Hfq stimulates the activity of the CCA-adding enzyme.

Scheibe M, Bonin S, Hajnsdorf E, Betat H, Mörl M - BMC Mol. Biol. (2007)

Bottom Line: Furthermore, Hfq binds specifically to tRNA transcripts, which seems to be the prerequisite for the observed effect on CCA-addition.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Biochemistry, University of Leipzig, Brüderstr, 34, 04103 Leipzig, Germany. mscheibe@uni-leipzig.de

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.

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Apparent kinetic parameters of CCA-addition. Increasing amounts of unlabeled tRNAPhe 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.
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Figure 3: Apparent kinetic parameters of CCA-addition. Increasing amounts of unlabeled tRNAPhe 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.

Mentions: To investigate the stimulating effect of Hfq on CCA-addition in more detail, Michaelis-Menten kinetic analyses were performed (Fig. 3, Table 1). In five independent experiments, apparent KM and Vmax values were determined in presence and absence of Hfq or BSA, respectively. Concentration of tRNAPhe without CCA end was ranging from 0.1 μM to 2.5 μM. Reaction products were separated by PAGE, and the signal intensities were quantified. While the presence of Hfq did not change KM values compared to reaction of the CCA-adding enzyme alone (0.36 μM versus 0.31 μM), it increases the maximum reaction rate of CCA-addition from 14.16 nM/min to 41.00 nM/min (Table 1). On the other hand, the stimulating effect of BSA had only a slight impact on Vmax, but lowered KM. This stimulating effect can be observed particularly at very low substrate concentrations (0.01 μM), where it seems to be equivalent to the Hfq-based reaction enhancement. At higher tRNA concentrations, however, BSA leads only to a slight increase in reaction velocity compared to the reaction in the presence of Hfq (Fig. 3).


Hfq stimulates the activity of the CCA-adding enzyme.

Scheibe M, Bonin S, Hajnsdorf E, Betat H, Mörl M - BMC Mol. Biol. (2007)

Apparent kinetic parameters of CCA-addition. Increasing amounts of unlabeled tRNAPhe 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175515&req=5

Figure 3: Apparent kinetic parameters of CCA-addition. Increasing amounts of unlabeled tRNAPhe 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.
Mentions: To investigate the stimulating effect of Hfq on CCA-addition in more detail, Michaelis-Menten kinetic analyses were performed (Fig. 3, Table 1). In five independent experiments, apparent KM and Vmax values were determined in presence and absence of Hfq or BSA, respectively. Concentration of tRNAPhe without CCA end was ranging from 0.1 μM to 2.5 μM. Reaction products were separated by PAGE, and the signal intensities were quantified. While the presence of Hfq did not change KM values compared to reaction of the CCA-adding enzyme alone (0.36 μM versus 0.31 μM), it increases the maximum reaction rate of CCA-addition from 14.16 nM/min to 41.00 nM/min (Table 1). On the other hand, the stimulating effect of BSA had only a slight impact on Vmax, but lowered KM. This stimulating effect can be observed particularly at very low substrate concentrations (0.01 μM), where it seems to be equivalent to the Hfq-based reaction enhancement. At higher tRNA concentrations, however, BSA leads only to a slight increase in reaction velocity compared to the reaction in the presence of Hfq (Fig. 3).

Bottom Line: Furthermore, Hfq binds specifically to tRNA transcripts, which seems to be the prerequisite for the observed effect on CCA-addition.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Biochemistry, University of Leipzig, Brüderstr, 34, 04103 Leipzig, Germany. mscheibe@uni-leipzig.de

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.

Show MeSH
Related in: MedlinePlus