Functional Escherichia coli 30S ribosomal subunits can be reconstituted in vitro. However, slow kinetics and sharp temperature dependence suggest additional assembly factors are present in vivo. Extract activation of in vitro assembly results in association of DnaK/hsp70 chaperone components with pre-30S particles. Purified DnaK, its cochaperones DnaJ and GrpE, and ATP can facilitate reconstitution of functional 30S subunits under otherwise nonpermissive conditions. A link has been observed between DnaK, 30S subunit components, and ribosome biogenesis in vivo as well as in vitro. These studies reveal a novel role for the DnaK/hsp70 chaperone system, in addition to its well-documented role in protein folding, and suggest that 30S subunit assembly can be facilitated.
Bibliographical noteFunding Information:
We thank J. Downing for his initial contributions to this work, A. Cukras for TP30 preparation, and K. Holmes for assistance with Figure 6 . R. Green and A. Andreotti are thanked for critical reading of the manuscript. We thank C. Sargenti and A. Fink for the DnaK-IMPACT plasmid and for guidance on DnaK overexpression, purification, and storage. This work is supported by National Institutes of Health Grant GM62432 to G.M.C.