Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

Abstract

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V O motor

“…The thickness of this detergent micelle is ∼25 Å, which is in good agreement with the known radius of a nonhydrated DDM micelle (45). Detergent has been observed previously in single particle cryo-EM maps, usually as blurring at the periphery of membrane proteins (e.g., 46), but to our knowledge the map presented here shows by far the most distinct delineation observed to date between protein and a detergent micelle in a cryo-EM structure of a membrane protein complex. To interpret the map further, we performed automated rigid-body fitting of available crystal structures and a comparative model of the L 12 − ring, either directly into the whole density map or into individual segmented densities.…”

Mapping of the Saccharomyces cerevisiae Oxa1-Mitochondrial Ribosome Interface and Identification of MrpL40, a Ribosomal Protein in Close Proximity to Oxa1 and Critical for Oxidative Phosphorylation Complex Assembly

“…This elegant study identified ribosomal proteins L23, L24, and L29 and the helix H59 of the 23S rRNA as physical contact points of YidC and the ribosome. Interestingly, these ribosomal contact points for YidC overlapped with those previously shown for the SecY translocon, although no direct sequence similarity exists between SecY and YidC (26). Overlapping contact points L23, L29, and H59 were observed with the Oxa1 protein and the E. coli ribosome; however, evidence for the L24 protein interacting with the Oxa1 protein was lacking.…”

The bacterial Sec-translocase: structure and mechanism

“…At the cytoplasmic face of the channel, several loops protrude from the membrane where they bind to cytosolic binding partners. Structural data show that loops between TMS 6/7 and TMS 8/9 form extensive contacts with the ribosome and SecA [30,45,46]. These observations are supported by cross-linking and mutagenesis studies where residues in these loops were shown to be important in translocation and/or binding of the cytoplasmic partner [46][47][48][49].…”

“…Structural data show that loops between TMS 6/7 and TMS 8/9 form extensive contacts with the ribosome and SecA [30,45,46]. These observations are supported by cross-linking and mutagenesis studies where residues in these loops were shown to be important in translocation and/or binding of the cytoplasmic partner [46][47][48][49]. The interaction of SecA and/or ribosomes with the SecYEG complex likely results in specific conformational changes of the channel, possibly even a partial opening as suggested by the SecASecYEG crystallographic structure.…”