Translocation complexes

Fig. 3. Topological coupling of DNA translocation and chromatin remodeling. (A) Alternative models for remodeling of a single nucleosome driven by the translocating complexes are compared with passive remodeling driven by the SP6 RNA polymerase or RNA polymerase III [109]. Note that no superhelicity would be constrained unless rotation of the translocase and DNA ends is impeded or prevented. It is also assumed that translocation occurs in steps of less than 5bp. CRA, chromatin remodelling assembly. The arrows indicate the direction of translocation of the DNA. (B) Model for remodeling of a nucleosome array within a topologically defined domain. Adapted with permission from Ref [119].

Proteins are targeted to various locations after synthesis by signal sequences. Thus, proteins destined for the ER, the mitochondria and chloroplasts have particular kinds of signal sequences at the N-terminus. ER-targeted proteins enter the ER directly off rough ER ribosomes via a signal recognition particle (SRP) complex that is linked to an SRP receptor and a ribosome receptor-transmembrane peptide translocation complex associated with the ER membrane. Within, the ER polypeptides are processed and folded and S—S links are formed. 

Reduction of the toxin and translocation of the L chain into the cytoplasm To gain access to the cytoplasm, the L chain needs to cross the membrane of the endocytic compartment. For this translocation, the H chain is required, probably by forming a proteinaceous translocation complex in the membrane that exposes (and possibly releases) the L chain to the cytoplasm. In the reductive intracellular environment, the disulfide bond linking the H and L chains is reduced (Kistner and Habermann, 1992). 

Feldheim, D., and Schekman, R. (1994). Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex./. CeUBiol. 126,... 

The ribosome receptor and translocation complex facilitate protein translocation into the lumen of the ER, where a signal peptidase cleaves the signal peptide. Both the SRP and ribosomal components are recycled back into the cytoplasm. 

Fig. 9. Maturation pathway of a class II non-lantibiotic bacteriocin presented as a 4-step process. The HPK and RR constitute a protein complex involved in signal transduction of external stimuli (step 1), resulting in transcription and translation of the bacteriocin precursor (step 2). The inactive precursor peptide is targeted to a membrane anchored ATP dependent translocation complex and processing at the double-glycine consensus site occurs intracellularly (step 3) and is followed by secretion (step 4). The role of the accessory protein in this mechanism is not yet understood... 

Kassenbrock, C.K., Cao, W. Douglas, M.G. (1993) EMBOJ., 12, 3023-3034. Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex. 

Now the question arises as to when and for what the fission of GTP is required. To solve this, we have studied the aminoacyl-tRNA binding reaction in the presence of a non-hydrolyzable analogue of GTP, Gpp(CH2)p or Gpp(NH)p. When a ribosomal complex having N-acetyl-P C]Phe-tRNA at the P site (Complex I, or post-translocational complex) was incubated with [ C]Phe-tRNA in the presence of EF-Tu-EF-Ts and GTP, a new complex (Complex II, or pretranslocational complex) was formed which possessed N-acetyl-di pC]Phe-tRNA and uncharged tRNA at the A and P sites, respectively. On the other hand, in experiments in which either Gpp(CH2)p or Gpp(NH)p was substituted for GTP, only negligible dipeptide formation was observed between the newly bound Phe-tRNA and the prebound N-acetyl-Phe-tRNA, and EF-Tu-Gpp(CH2)p or EF-Tu-Gpp-(NH)p remained bound on ribosomes. This suggested that the hydrolysis of GTP is required in some step prior to the formation of the new peptide bond. 

For these studies we formed an initiation complex on f2 RNA, converted an aliquot into a pretranslocation complex by reacting it with the appropriate EF-Tu GTP aminoacyl-tRNA complex. An aliquot of the pretranslocation complex was then converted into a post-translocation complex by treatment with EF-G and GTP. After treating each of the complexes with pancreatic ribonuclease, we sequenced the protected f2 RNA segment in each. The 3 end of the protected segment was the same in the initiation and pretranslocation complexes it extended three nucleotides further toward the 3 end in the posttranslocation complex. In accord with these results, the translation of the protected f2 RNA segments from the initiation and pretranslocation complexes resulted in the same pentapeptide, that from the post-translocation complex in the expected hexapeptide. These findings proved that the ribosome movement occurs during translocation and depends on EF-G and GTP. 

Figure I. The glycosylation reaction catalyzed by oligosaccharyltransferase. The unique core oligosaccharide Glc3Man9GlcNAc2 is transferred from dolichyl pyrophosphate to selected asparagine residues in an Asn-A-Ser/Thr consensus sequon of nascent polypeptides. The dolichol moiety contains an saturated a-isoprenoid unit and may vary in chain length. The OST complex from yeast is shown. Subunits in bold face are essential for yeast growth SEC61 represents the yeast protein translocation complex.

DNP enhancement was also used to investigate 40 nmol of a 25-residue signal peptide bound to the lipid-reconstituted 600-residue protein translocation complex SecY translocon [229]. Although double quantum filtering was mandatory to suppress the large natural abundance SecY background, a decent 2D-spectrum of the peptide could be obtained within 20 h of measurement time. For three of four isotope labeled amino acids the spin system could be identified by a sequential walk. The corresponding secondary chemical shifts were indicative of an a-helical secondary structure of the peptide in its bound form. 

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