A basic region neighboring the lysine-rich C-terminus of protein SRP19 is required for binding to signal recognition particle RNA

1991 ◽  
Vol 69 (9) ◽  
pp. 649-654 ◽  
Author(s):  
Christian Zwieb
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Signal Recognition Particle ◽  
Site Directed Mutagenesis ◽  
Signal Recognition ◽  
C Terminus ◽  
Terminal Amino ◽  
Rna Complexes ◽  
Mutant Polypeptides

To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein–RNA complexes on DEAE–Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine-rich sequence at positions 136–144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113–120; they are suggested to be involved in interaction with signal recognition particle RNA.Key words: signal recognition particle, site-directed mutagenesis, protein–RNA binding.

scholarly journals The 54-kD protein of signal recognition particle contains a methionine-rich RNA binding domain.

1990 ◽  
Vol 111 (5) ◽  
pp. 1793-1802 ◽  
Author(s):  
K Römisch ◽  
J Webb ◽  
K Lingelbach ◽  
H Gausepohl ◽  
B Dobberstein
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Binding Domain ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Rna Binding Domain ◽  
Necessary And Sufficient

Signal recognition particle (SRP) plays the key role in targeting secretory proteins to the membrane of the endoplasmic reticulum (Walter, P., and V. R. Lingappa. 1986. Annu. Rev. Cell Biol. 2:499-516). It consists of SRP7S RNA and six proteins. The 54-kD protein of SRP (SRP54) recognizes the signal sequence of nascent polypeptides. The 19-kD protein of SRP (SRP19) binds to SRP7S RNA directly and is required for the binding of SRP54 to the particle. We used deletion mutants of SRP19 and SRP54 and an in vitro assembly assay in the presence of SRP7S RNA to define the regions in both proteins which are required to form a ribonucleoprotein particle. Deletion of the 21 COOH-terminal amino acids of SRP19 does not interfere with its binding to SRP7S RNA. Further deletions abolish SRP19 binding to SRP7S RNA. The COOH-terminal 207 amino acids of SRP54 (M domain) were found to be necessary and sufficient for binding to the SRP19/7S RNA complex in vitro. Limited protease digestion of purified SRP confirmed our results for SRP54 from the in vitro binding assay. The SRP54M domain could also bind to Escherichia coli 4.5S RNA that is homologous to part of SRP7S RNA. We suggest that the methionine-rich COOH terminus of SRP54 is a RNA binding domain and that SRP19 serves to establish a binding site for SRP54 on the SRP7S RNA.

IMPROVEMENT OF T-PA PROPERTIES BY MEANS OF SITE DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
N Haigwood ◽  
E-P Pâques ◽  
G Mullenbach ◽  
G Moore ◽  
L DesJardin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Specific Activity ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Proteins ◽  
C Terminus

The clinical relevance of tissue-plasminogen-activator (t-PA) as a potent thrombolytic agent has recently been established. It has however been recognized that t-PA does not fulfill all conditions required for an ideal thrombolytic pharmaceutical agent; for example, its physiological stability and its short half life in vivo necessitate the use of very large clinical doses. We have therefore attempted to develop novel mutant t-PA proteins with improved properties by creating mutants by site-directed mutagenesis in M13 bacteriophage. Seventeen mutants were designed, cloned, and expressed in CHO cells. Modifications were of three types: alterations to glycosylation sites, truncations of the N- or C-termini, and amino acids changes at the cleavage site utilized to generate the two chain form of t-PA. The mutant proteins were analyzed in vitro for specific activity, fibrin dependence of the plasminogen activation, fibrin affinity, and susceptibility to inhibition by PAI.In brief, the results are: 1) some unglycosylated and partially glycosylated molecules obtained by mutagenesis are characterized by several-fold higher specific activity than wild type t-PA; 2) truncation at the C-terminus by three amino acids yields a molecule with increased fibrin specificity; 3) mutations at the cleavage site lead zo a decreased inhibition by PAI; and 4) recombinants of these genes have been constructed and the proteins were shown to possess multiple improved properties. The use of site directed mutagenesis has proved to be a powerful instrument to modulate the biological properties of t-PA.

scholarly journals The DEAD-box RNA helicase Ded1 from yeast is associated with the signal recognition particle (SRP), and its enzymatic activity is regulated by SRP21

2020 ◽  
Author(s):  
Hilal Yeter-Alat ◽  
Naïma Belgareh-Touzé ◽  
Emmeline Huvelle ◽  
Molka Mokdadi ◽  
Josette Banroques ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Rna Binding ◽  
Signal Recognition Particle ◽  
Rna Helicase ◽  
Signal Recognition ◽  
Dead Box ◽  
Enzymatic Regulation ◽  
The Dead

ABSTRACTThe DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation. It belongs to the DDX3 subfamily of proteins implicated in developmental and cell-cycle regulation. In vitro, the purified Ded1 protein is an ATP-dependent RNA binding protein and an RNA-dependent ATPase, but it lacks RNA substrate specificity and enzymatic regulation. Here we demonstrate by yeast genetics, in situ localization and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 with SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results open a new comprehension of the cellular role of Ded1 during translation.

scholarly journals Association of Influenza Virus Matrix Protein with Ribonucleoproteins

1999 ◽  
Vol 73 (9) ◽  
pp. 7467-7473 ◽  
Author(s):  
Zhiping Ye ◽  
Teresa Liu ◽  
Daniel P. Offringa ◽  
Jonathan McInnis ◽  
Roland A. Levandowski
Keyword(s):  
Amino Acids ◽  
Zinc Finger ◽  
Influenza A ◽  
Matrix Protein ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Zinc Finger Motif ◽  
Basic Amino Acids ◽  
Binding Regions

ABSTRACT To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.

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 Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

2009 ◽  
Vol 84 (3) ◽  
pp. 1406-1413 ◽  
Author(s):  
Brian C. Lin ◽  
Dawn A. Defenbaugh ◽  
John L. Casey
Keyword(s):  
Conformational Changes ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Minimum Length ◽  
Critical Determinant ◽  
Hepatitis Delta ◽  
Ribonucleoprotein Complex ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen

ABSTRACT Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.

scholarly journals Anionic amino acids support hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

2020 ◽  
pp. jbc.RA120.015524
Author(s):  
Alexandra P Breslawec ◽  
Shaochi Wang ◽  
Crystal Li ◽  
Myles B Poulin
Keyword(s):  
Amino Acids ◽  
Bacterial Infections ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Binding Surface ◽  
Activity Measurements ◽  
Rate Of Hydrolysis ◽  
Biofilm Dispersal ◽  
Hydrolysis Of

The exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity on de-N-acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the –1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

scholarly journals The CELF1 RNA-Binding Protein Regulates Decay of Signal Recognition Particle mRNAs and Limits Secretion in Mouse Myoblasts

PLoS ONE ◽  
2017 ◽  
Vol 12 (1) ◽  
pp. e0170680 ◽  
Author(s):  
Joseph Russo ◽  
Jerome E. Lee ◽  
Carolina M. López ◽  
John Anderson ◽  
Thuy-mi P. Nguyen ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Signal Recognition Particle ◽  
Rna Binding Protein ◽  
Signal Recognition

Coronin localizes to leading edges and is involved in cell spreading and lamellipodium extension in vertebrate cells

1999 ◽  
Vol 112 (17) ◽  
pp. 2833-2842 ◽  
Author(s):  
M. Mishima ◽  
E. Nishida
Keyword(s):  
Amino Acids ◽  
Cell Spreading ◽  
Immunofluorescent Staining ◽  
Actin Binding ◽  
Cellular Slime Mold ◽  
Cell Periphery ◽  
Terminal Amino ◽  
Wd Repeat ◽  
Leading Edges

Coronin is a WD repeat-containing actin-binding protein, which was originally identified in the cellular slime mold Dictyostelium. Coronin-null Dictyostelium cells show defects in cytokinesis, cell motility and phagocytosis. Although the existence of coronin in higher eukaryotes has been reported, its function in vertebrate cells has not been elucidated. We cloned a Xenopus homolog of coronin (Xcoronin) and examined its actin-binding properties, subcellular localization and possible functions. Xcoronin consists of 480 amino acids and is 63% identical to human coronin (p57). Bacterially expressed recombinant Xcoronin co-sedimented with F-actin in vitro. The WD repeat domain (residues 64–299) alone did not have any affinity for F-actin. Anti-Xcoronin antibodies reacted specifically with a single 57 kDa protein present in an extract of the Xenopus A6 cell line. Indirect immunofluorescent staining of A6 cells revealed that Xcoronin is present in the cytoplasm and concentrated in the cell periphery in membrane ruffles. During spreading after replating or wound healing after scratching a confluent monolayer, Xcoronin became concentrated in the leading edges of lamellipodia. A GFP-fusion protein of Xcoronin showed a subcellular distribution essentially identical to endogenous Xcoronin. The localization of Xcoronin to the cell periphery was resistant to treatment with 0.1% Triton X-100. The deletion of 63 N-terminal amino acids or of 65 C-terminal amino acids abolished the localization of Xcoronin to the cell periphery. Xcoronin expressed in 3T3 fibroblasts was concentrated to the leading edges of lamellipodia induced by active Rac. Remarkably, expression of a truncated form of Xcoronin (64–299), but not of full-length Xcoronin, significantly decreased the rate of cell spreading after replating and markedly inhibited lamellipodium extension induced by active Rac. These results suggest that Xcoronin plays an important role in lamellipodium extension and cell spreading.

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