The Cost of Exposing a Hydrophobic Loop and Implications for the Functional Role of 4.5 S RNA in the Escherichia coli Signal Recognition Particle (original) (raw)
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Structure of the E. coli signal recognition particle bound to a translating ribosome
Nature, 2007
The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane 1-4 . It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 Å structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.
RNA
The structure of 4.5S RNA, the Escherichia coli homologue of the signal recognition particle (SRP) RNA, alone and in the SRP complex with protein P48 (Ffh) was probed both enzymatically and chemically. The molecule is largely resistant against single strand-specific nucleases, indicating a highly base paired structure. Reactivity appears mainly in the apical tetraloop and in one of the conserved internal loops. Although some residues are found reactive toward dimethylsulphate and kethoxal in regions predicted to be unpaired by the phylogenetic secondary structure model of 4.5S RNA, generally the reactivity is low, and some residues in internal loops are not reactive at all. RNase V1 cleaves the RNA at multiple sites that coincide with predicted helices, although the cleavages show a pronounced asymmetry. The binding of protein P48 to 4.5S RNA results in a protection of residues in the apical part of the molecule homologous to eukaryotic SRP RNA (domain IV), whereas the cleavages in ...
RNA, 2003
The signal recognition particle (SRP) from Escherichia coli, composed of Ffh protein and 4.5S RNA, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence. SRP binds at the peptide exit of the large ribosomal subunit. Structural details of the interaction are not known. Here, the position of Ffh or SRP on the ribosome was probed by using site-specific UV-induced crosslinking by p-azidophenacyl bromide (AzP) attached to a number of cysteine residues engineered into surface positions of Ffh. Efficient crosslinking to vacant ribosomes took place from two positions (AzP17 and AzP25) in the N domain of Ffh, both with Ffh and SRP. Both AzP17 and AzP25 were predominantly crosslinked to ribosomal protein L23 that is located at the peptide exit of the 50S subunit. The SRP receptor, FtsY, did not change the crosslink pattern, whereas the presence of a nascent signal peptide on the ribosome resulted in a second crosslink between Ffh(AzP17) and protein L23, indicating that binding to the nascent signal peptide induced a slightly different arrangement of SRP on the ribosome. These results indicate a model of the topographical arrangement of SRP at the peptide exit of the 50S ribosomal subunit.
FEBS Letters, 2002
Escherichia coli signal recognition particle (SRP) consists of 4.5S RNA and Ffh protein. In contrast to eukaryotes, it remains unclear whether translation arrest takes place in prokaryotic cells. To study this problem we constructed a fusion of the M domain of Ffh protein with a cleavable affinity tag. This mutant Ffh, in a complex with 4.5S RNA, can bind signal peptide at the translating ribosome but is unable to bind the membrane. This SRP^ribosome complex should accumulate in the cell if translation is arrested. To test this, the complex was purified from the cells by ultracentrifugation and affinity chromatography. The composition of the complex was analyzed and found to consist of ribosomal RNAs and proteins, the Ffh M domain and 4.5S RNA. The accumulation of this complex in the cell in significant amounts indicated that SRP-mediated translation arrest did occur in bacterial cells.
The Journal of Cell Biology, 2003
As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro–synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The...
Molecular Biology of the Cell, 2006
Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for E. coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data was fit into the SRP reconstruction and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances consistent with our model of SRP.