scholarly journals Stabilizing Obligatory Non-native Intermediates Along Co-transcriptional Folding Trajectories of SRP RNA Affects Cell Viability

2018 ◽  
Author(s):  
Shingo Fukuda ◽  
Shannon Yan ◽  
Yusuke Komi ◽  
Mingxuan Sun ◽  
Ronen Gabizon ◽  
...  
Keyword(s):  
Cell Viability ◽  
Optical Tweezers ◽  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Folding Pathway ◽  
List Type ◽  
Transcription Complexes ◽  
Native Fold ◽  
Srp Rna

SummarySignal recognition particle (SRP) inEscherichia colicomprises protein Ffh and SRP RNA. Its essential functionality—co-translational protein-targeting/delivery to cellular membranes— hinges on the RNA attaining a native long-hairpin fold that facilitates protein conformational rearrangements within the SRP complex. Since RNA folds co-transcriptionally on RNA polymerase, we use high-resolution optical tweezers to first characterize the mechanical unfolding/refolding of incrementally lengthened RNAs from stalled transcription complexes until reaching the full-length transcript. This analysis allows identification of folding intermediates adopted during the real-time co-transcriptional folding of SRP RNA. The co-transcriptional folding trajectories are surprisingly invariant to transcription rates, and involve formation of an obligatory non-native hairpin intermediate that eventually resolves into the native fold. SRP RNA variants designed to stabilize this non-native intermediate—likely sequestering the SRP ribonucleoprotein complex in an inactive form—greatly reduce cell viability, indicating that the same co-transcriptional folding mechanism operatesin vivoand possible alternative antibiotic strategies.HighlightsFolding pathway of an essential functional RNA has been resolved co-transcriptionally.The co-transcriptional folding pathway of SRP RNA is invariant to transcription rates.Nascent SRP RNA obligatorily forms a non-native intermediate before adopting the native fold.Modulating transitions from the non-native to native SRP RNA hairpin fold alters cell viability.

scholarly journals Computationally Reconstructing Cotranscriptional RNA Folding Pathways from Experimental Data Reveals Rearrangement of Non-Native Folding Intermediates

2018 ◽  
Author(s):  
Angela M Yu ◽  
Paul M. Gasper ◽  
Eric J. Strobel ◽  
Kyle E. Watters ◽  
Alan A. Chen ◽  
...  
Keyword(s):  
Rna Folding ◽  
Signal Recognition Particle ◽  
Point Mutations ◽  
Structural Rearrangement ◽  
Folding Pathway ◽  
List Type ◽  
Folding Intermediates ◽  
Folding Pathways ◽  
Dynamics Simulations ◽  
Srp Rna

SummaryThe series of RNA folding events that occur during transcription, or a cotranscriptional folding pathway, can critically influence the functional roles of RNA in the cell. Here we present a method, Reconstructing RNA Dynamics from Data (R2D2), to uncover details of cotranscriptional folding pathways by predicting RNA secondary and tertiary structures from cotranscriptional SHAPE-Seq data. We applied R2D2 to the folding of the Escherichia coli Signal Recognition Particle (SRP) RNA sequence and show that this sequence undergoes folding through non-native intermediate structures that require significant structural rearrangement before reaching the functional native structure. Secondary structure folding pathway predictions and all-atom molecular dynamics simulations of folding intermediates suggest that this rearrangement can proceed through a toehold mediated strand displacement mechanism, which can be disrupted and rescued with point mutations. These results demonstrate that even RNAs with simple functional folds can undergo complex folding processes during synthesis, and that small variations in their sequence can drastically affect their cotranscriptional folding pathways.Highlights- Computational methods predict RNA structures from cotranscriptional SHAPE-Seq data- The E. coli SRP RNA folds into non-native structural intermediates cotranscriptionally- These structures rearrange dynamically to form an extended functional fold- Point mutations can disrupt and rescue cotranscriptional RNA folding pathways

scholarly journals The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting

2007 ◽  
Vol 18 (7) ◽  
pp. 2728-2734 ◽  
Author(s):  
Niels Bradshaw ◽  
Peter Walter
Keyword(s):  
Conformational Changes ◽  
Protein Targeting ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Gtp Hydrolysis ◽  
E Coli ◽  
Srp Rna ◽  
Binding Subunit

The RNA component of the signal recognition particle (SRP) is universally required for cotranslational protein targeting. Biochemical studies have shown that SRP RNA participates in the central step of protein targeting by catalyzing the interaction of the SRP with the SRP receptor (SR). SRP RNA also accelerates GTP hydrolysis in the SRP·SR complex once formed. Using a reverse-genetic and biochemical analysis, we identified mutations in the E. coli SRP protein, Ffh, that abrogate the activity of the SRP RNA and cause corresponding targeting defects in vivo. The mutations in Ffh that disrupt SRP RNA activity map to regions that undergo dramatic conformational changes during the targeting reaction, suggesting that the activity of the SRP RNA is linked to the major conformational changes in the signal sequence-binding subunit of the SRP. In this way, the SRP RNA may coordinate the interaction of the SRP and the SR with ribosome recruitment and transfer to the translocon, explaining why the SRP RNA is an indispensable component of the protein targeting machinery.

SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation

Science ◽  
2014 ◽  
Vol 344 (6179) ◽  
pp. 101-104 ◽  
Author(s):  
Jan Timo Grotwinkel ◽  
Klemens Wild ◽  
Bernd Segnitz ◽  
Irmgard Sinning
Keyword(s):  
Ribosomal Rna ◽  
Protein Targeting ◽  
Rna Binding ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Structural Basis ◽  
Signal Recognition ◽  
Rna Remodeling ◽  
Srp Rna ◽  
Arginine Rich Motif

The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

scholarly journals In Vivo Analysis of an Essential Archaeal Signal Recognition Particle in Its Native Host

2002 ◽  
Vol 184 (12) ◽  
pp. 3260-3267 ◽  
Author(s):  
R. Wesley Rose ◽  
Mechthild Pohlschröder
Keyword(s):  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Sequence Comparisons ◽  
Protein Insertion ◽  
Native Host

ABSTRACT The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.

scholarly journals Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

2015 ◽  
Vol 112 (13) ◽  
pp. 3943-3948 ◽  
Author(s):  
Ottilie von Loeffelholz ◽  
Qiyang Jiang ◽  
Aileen Ariosa ◽  
Manikandan Karuppasamy ◽  
Karine Huard ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Targeting ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Binding Pocket ◽  
Van Der Waals Interactions ◽  
Closed State ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Srp Rna

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome–nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP–RNC complex results in GTP-independent binding of the SRP–FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP–FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP–FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.

scholarly journals Structure, dynamics and interactions of large SRP variants

2019 ◽  
Vol 401 (1) ◽  
pp. 63-80 ◽  
Author(s):  
Klemens Wild ◽  
Matthias M.M. Becker ◽  
Georg Kempf ◽  
Irmgard Sinning
Keyword(s):  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Domain Architecture ◽  
Particle Dynamics ◽  
Signal Recognition ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Target Membrane ◽  
Signal Anchor ◽  
Srp Rna

Abstract Co-translational protein targeting to membranes relies on the signal recognition particle (SRP) system consisting of a cytosolic ribonucleoprotein complex and its membrane-associated receptor. SRP recognizes N-terminal cleavable signals or signal anchor sequences, retards translation, and delivers ribosome-nascent chain complexes (RNCs) to vacant translocation channels in the target membrane. While our mechanistic understanding is well advanced for the small bacterial systems it lags behind for the large bacterial, archaeal and eukaryotic SRP variants including an Alu and an S domain. Here we describe recent advances on structural and functional insights in domain architecture, particle dynamics and interplay with RNCs and translocon and GTP-dependent regulation of co-translational protein targeting stimulated by SRP RNA.

Identification of RNA sequences and structural elements required for assembly of fission yeast SRP54 protein with signal recognition particle RNA

1993 ◽  
Vol 13 (3) ◽  
pp. 1353-1362
Author(s):  
D Selinger ◽  
P Brennwald ◽  
X Liao ◽  
J A Wise
Keyword(s):  
Fission Yeast ◽  
Base Pair ◽  
Structural Integrity ◽  
Signal Recognition Particle ◽  
Yeast Cells ◽  
Growth Defect ◽  
Signal Recognition ◽  
Nucleotide Substitutions ◽  
Srp Rna

Signal recognition particle (SRP) is a ribonucleoprotein composed of six polypeptides and a single RNA molecule. SRP RNA can be divided into four structural domains, the last of which is the most highly conserved and, in Schizosaccharomyces pombe, is the primary location to which deleterious mutations map. The ability of mammalian SRP54 protein (SRP54p) to bind Escherichia coli 4.5S RNA, a homolog of SRP RNA which contains only domain IV, suggested that SRP54p might interact directly with this region. To determine whether domain IV is critical for SRP54p binding in fission yeast cells, we used a native immunoprecipitation-RNA sequencing assay to test 13 mutant SRP RNAs for the ability to associate with the protein in vivo. The G156A mutation, which alters the 5' residue of the noncanonical first base pair of the domain IV terminal helix and confers a mild conditional growth defect, reduces assembly of the RNA with SRP54p. Mutating either of the two evolutionarily invariant residues in the bulged region 5' to G156 is more deleterious to growth and virtually abolishes SRP54p binding. We conclude that the conservation of nucleotides 154 to 156 is likely to be a consequence of their role as a sequence-specific recognition element for the SRP54 protein. We also tested a series of mutants with nucleotide substitutions in the conserved tetranucleotide loop and adjoining stem of domain IV. Although tetraloop mutations are deleterious to growth, they have little effect on SRP54p binding. Mutations which disrupt the base pair flanking the tetraloop result in conditional growth defects and significantly reduce association with SRP54p. Disruption of the other two base pairs in the short stem adjacent to the tetranucleotide loop has similar but less dramatic effects on SRP54p binding. These data provide the first evidence that both sequence-specific contacts and the structural integrity of domain IV of SRP RNA are important for assembly with SRP54p.

scholarly journals Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae.

1992 ◽  
Vol 3 (8) ◽  
pp. 895-911 ◽  
Author(s):  
S C Ogg ◽  
M A Poritz ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Yeast Cells ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Er Membrane

In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent.

scholarly journals A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

2009 ◽  
Vol 20 (17) ◽  
pp. 3965-3973 ◽  
Author(s):  
Peera Jaru-Ampornpan ◽  
Thang X. Nguyen ◽  
Shu-ou Shan
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Receptor Interaction ◽  
Signal Recognition ◽  
Gtpase Domain ◽  
Distinct Mechanism ◽  
Molecular Origin ◽  
Srp Rna

Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5- to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication.

scholarly journals Defining the physiological role of SRP in protein-targeting efficiency and specificity

Science ◽  
2018 ◽  
Vol 359 (6376) ◽  
pp. 689-692 ◽  
Author(s):  
Elizabeth A. Costa ◽  
Kelly Subramanian ◽  
Jodi Nunnari ◽  
Jonathan S. Weissman
Keyword(s):  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Physiological Role ◽  
Ribosome Profiling ◽  
Signal Recognition ◽  
Amino Terminal ◽  
Mitochondrial Defects ◽  
Unanticipated Consequence

The signal recognition particle (SRP) enables cotranslational delivery of proteins for translocation into the endoplasmic reticulum (ER), but its full in vivo role remains incompletely explored. We combined rapid auxin-induced SRP degradation with proximity-specific ribosome profiling to define SRP’s in vivo function in yeast. Despite the classic view that SRP recognizes amino-terminal signal sequences, we show that SRP was generally essential for targeting transmembrane domains regardless of their position relative to the amino terminus. By contrast, many proteins containing cleavable amino-terminal signal peptides were efficiently cotranslationally targeted in SRP’s absence. We also reveal an unanticipated consequence of SRP loss: Transcripts normally targeted to the ER were mistargeted to mitochondria, leading to mitochondrial defects. These results elucidate SRP’s essential roles in maintaining the efficiency and specificity of protein targeting.

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