Structure of the E. coli protein-conducting channel bound to a translating ribosome

Abstract

Secreted and membrane proteins are translocated across/into cell membranes via a proteinconducting channel (PCC). We present a cryo-EM reconstruction of the E. coli PCC, SecYEG, complexed with the ribosome and a signal anchor containing nascent chain, showing mRNA, three tRNAs, the nascent chain, and detailed features of both a translocating PCC and a second, nontranslocating PCC bound to mRNA hairpins. The translocating PCC forms connections with ribosomal RNA hairpins on two sides and ribosomal proteins at the back, leaving a frontal opening. Normal mode-based flexible fitting of the archaeal SecYEβ structure into the PCC EM densities favors a front-to-front arrangement of two SecYEG complexes in the PCC, and supports channel formation by the opening of two linked SecY halves during polypeptide translocation. Based on our observation in the translocating PCC of two segregated pores with different degrees of access to bulk lipid, we propose a model for co-translational protein translocation.

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

“…A similar mechanism could underlie the ribosome induced opening mechanism as well, as the ribosome also interacts with the C5 loop 37 and with the cytoplasmic domain of SecG which is bound to the Nterminal domain of SecY. 14 Taken together, our studies provide the first demonstration of two SecY regions that interact with SecA, including a region where SecA possibly inserts into the SecYEG channel. In addition, our data provide a framework that couples conformational changes in SecA to the previously proposed opening mechanism of the SecYEG channel.…”

“…The last five years have been characterized by a tremendous progress in our structural view on protein translocation, by the appearance of high resolution crystal structures of individual components [6][7][8][9][10][11][12] and medium resolution cryo-electron microscopy (cryo-EM) structures of co-translational translocation intermediates. 13,14 Despite the availability of these structures, there remains a large gap in our understanding of the interactions between the individual components, most notably on the highly dynamic interaction between SecYEG and SecA.…”

Dimeric organization of the yeast oligosaccharyl transferase complex

“…However, we note that the actively translocating translocon was recently determined to form a dimer on ribosomes (40). Because OT is known to physically associate with and to function in collaboration with the translocon (41), we speculate that a dimeric configuration of the OT complex may be required for effective association with the translocon dimer and efficient N-glycosylation of newly synthesized polypeptides.…”

Solution structure of RNase P RNA

“…MD simulations are highly sensitive to, and restricted by, the starting conformation, and so may fail to capture significant motions in macromolecular models based primarily on homology and biochemical information, e.g., the Eco and Atu RNA models described above. Since such collective motions in macromolecules often correlate with perturbations along elastic normal modes (Delarue and Sanejouand 2002;Tama et al 2003;Mitra et al 2005; Matsumoto and Ishida 2009), we investigated whether coarse-grain normal mode analysis (NMA) (Tirion 1996) combined with the ensemble optimization method (Bernado et al 2007) could extract structural models of these RNase P RNAs with improved fit to the SAXS data.…”