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).