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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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CCA-addition is stimulated by Hfq. (A) The E. coli CCA-adding enzyme was incubated for indicated times with radioactively labeled yeast tRNAPhe 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 tRNAAla, phage T5 tRNACys, 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).
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Figure 1: CCA-addition is stimulated by Hfq. (A) The E. coli CCA-adding enzyme was incubated for indicated times with radioactively labeled yeast tRNAPhe 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 tRNAAla, phage T5 tRNACys, 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).

Mentions: To investigate whether Hfq has an effect on the catalytic activity of the E. coli CCA-adding enzyme, the recombinant enzyme was incubated at 30°C for up to 10 minutes in the presence of NTPs and Hfq. As a radioactively labeled substrate for CCA-addition, yeast tRNAPhe lacking the CCA terminus was used. This is one of the best characterized tRNA molecules, and the corresponding unmodified in vitro transcript has a three-dimensional structure very similar to that of the fully modified in vivo molecule [20-23]. It is therefore an ideal substrate for in vitro tRNA processing and aminoacylation reactions [24-26]. In control experiments, addition of Hfq was omitted or replaced by bovine serum albumin (BSA), which is frequently used to stabilize purified enzymes in an active conformation. Furthermore, additional prokaryotic RNA binding proteins (E. coli NusA [27], Z. mobilis tRNA guanine transglycosilase (TGT) [28], E. coli RNase P protein [29] and E. coli HU [30,31]) as well as two variants of Hfq were used as controls. Hfq K56A, located in the highly conserved cavity of the hexamer, interferes with binding of the small regulatory RNA DsrA [32]. Hfq V43R, on the other hand, affects binding of polyadenylated rpsO mRNA and stimulation of PAP [33]. The reaction products were separated on polyacrylamide gels and visualized by autoradiography. As shown in Fig. 1A, CCA-addition leads to new tRNA species with reduced electrophoretic mobility, where the uppermost signal corresponds to the tRNA carrying a complete CCA terminus, while the other products indicate partial CCA-addition (incorporation of one or two C residues, respectively). While this activity is found in all assays, the time course indicates that Hfq enhances the reaction efficiency substantially. After 3 minutes of incubation, a strong increase of the reaction product (tRNAPhe with CCA terminus; Fig. 1A) can be seen in the reaction assay including Hfq, while the presence of BSA leads only to a moderate product increase relative to the reaction catalyzed by the CCA-adding enzyme alone. At longer incubation times, the reactions follow this trend. However, this stimulation is not the result of an Hfq- or BSA-catalyzed nucleotide incorporation, since incubation of tRNA substrate with NTPs and Hfq or BSA alone does not lead to any detectable reaction product (data not shown). The results were verified in 8 independent experiments. Reproductions of this assay with other tRNA transcripts without CCA end (E. coli tRNAAla, phage T5 tRNACys) further corroborated the general Hfq-mediated enhancement of CCA-addition (data not shown). The additional control experiments with RNA binding proteins and Hfq variants shown in Fig. 1B support these findings. None of the RNA binding proteins shows a stimulation of CCA-addition comparable to that of Hfq, indicating that this enhancement is indeed a specific effect of this protein. Furthermore, the fact that both Hfq variants stimulate CCA-addition comparable to the wild type protein demonstrates that this effect is different from PAP stimulation or interaction with small regulatory RNAs, where these point mutations lead to reduced efficiencies [32,33].


Hfq stimulates the activity of the CCA-adding enzyme.

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

CCA-addition is stimulated by Hfq. (A) The E. coli CCA-adding enzyme was incubated for indicated times with radioactively labeled yeast tRNAPhe 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 tRNAAla, phage T5 tRNACys, 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: CCA-addition is stimulated by Hfq. (A) The E. coli CCA-adding enzyme was incubated for indicated times with radioactively labeled yeast tRNAPhe 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 tRNAAla, phage T5 tRNACys, 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).
Mentions: To investigate whether Hfq has an effect on the catalytic activity of the E. coli CCA-adding enzyme, the recombinant enzyme was incubated at 30°C for up to 10 minutes in the presence of NTPs and Hfq. As a radioactively labeled substrate for CCA-addition, yeast tRNAPhe lacking the CCA terminus was used. This is one of the best characterized tRNA molecules, and the corresponding unmodified in vitro transcript has a three-dimensional structure very similar to that of the fully modified in vivo molecule [20-23]. It is therefore an ideal substrate for in vitro tRNA processing and aminoacylation reactions [24-26]. In control experiments, addition of Hfq was omitted or replaced by bovine serum albumin (BSA), which is frequently used to stabilize purified enzymes in an active conformation. Furthermore, additional prokaryotic RNA binding proteins (E. coli NusA [27], Z. mobilis tRNA guanine transglycosilase (TGT) [28], E. coli RNase P protein [29] and E. coli HU [30,31]) as well as two variants of Hfq were used as controls. Hfq K56A, located in the highly conserved cavity of the hexamer, interferes with binding of the small regulatory RNA DsrA [32]. Hfq V43R, on the other hand, affects binding of polyadenylated rpsO mRNA and stimulation of PAP [33]. The reaction products were separated on polyacrylamide gels and visualized by autoradiography. As shown in Fig. 1A, CCA-addition leads to new tRNA species with reduced electrophoretic mobility, where the uppermost signal corresponds to the tRNA carrying a complete CCA terminus, while the other products indicate partial CCA-addition (incorporation of one or two C residues, respectively). While this activity is found in all assays, the time course indicates that Hfq enhances the reaction efficiency substantially. After 3 minutes of incubation, a strong increase of the reaction product (tRNAPhe with CCA terminus; Fig. 1A) can be seen in the reaction assay including Hfq, while the presence of BSA leads only to a moderate product increase relative to the reaction catalyzed by the CCA-adding enzyme alone. At longer incubation times, the reactions follow this trend. However, this stimulation is not the result of an Hfq- or BSA-catalyzed nucleotide incorporation, since incubation of tRNA substrate with NTPs and Hfq or BSA alone does not lead to any detectable reaction product (data not shown). The results were verified in 8 independent experiments. Reproductions of this assay with other tRNA transcripts without CCA end (E. coli tRNAAla, phage T5 tRNACys) further corroborated the general Hfq-mediated enhancement of CCA-addition (data not shown). The additional control experiments with RNA binding proteins and Hfq variants shown in Fig. 1B support these findings. None of the RNA binding proteins shows a stimulation of CCA-addition comparable to that of Hfq, indicating that this enhancement is indeed a specific effect of this protein. Furthermore, the fact that both Hfq variants stimulate CCA-addition comparable to the wild type protein demonstrates that this effect is different from PAP stimulation or interaction with small regulatory RNAs, where these point mutations lead to reduced efficiencies [32,33].

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