3.6.5.4 A142W site-directed mutagenesis, the mutant cpSRP54 exhibits the same GTP-dependent complex assembly kinetics as wild-type cpSRP54 718911 3.6.5.4 A143L site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview 718911 3.6.5.4 A143W mutant, GTPase activation defective 687905 3.6.5.4 A143W site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview 718911 3.6.5.4 A144W mutant, GTPase activation defective 687905 3.6.5.4 A145S substitution introduced into the putative GTP binding motif GXXGXGK 684117 3.6.5.4 A168W site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview 718911 3.6.5.4 A169L site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview 718911 3.6.5.4 A169W site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview 718911 3.6.5.4 A192D reduced GTP hydrolysis, no effect on the interaction with FtsY 658538 3.6.5.4 A254L no growth defect -, 658538 3.6.5.4 A334W mutant, exhibits no significant GTP hydrolysis 687902 3.6.5.4 A335W mutant, GTPase activation defective 687905 3.6.5.4 A336W mutant, GTPase activation defective 687905 3.6.5.4 C86A mutations at C86 yield a more complex pattern: whereas C86A and C86U completely abolish GTPase activation by the RNA, C86 and C86G reduce GTPase activity by only 50% 734245 3.6.5.4 C86G mutations at C86 yield a more complex pattern: whereas C86A and C86U completely abolish GTPase activation by the RNA, DELTAC86 and C86G reduce GTPase activity by only 50%. Despite defective GTP hydrolysis, the G83A mutant shows any detectable defect in the efficiency of GTPase docking at the distal end 734245 3.6.5.4 C86U mutations at C86 yield a more complex pattern: whereas C86A and C86U completely abolish GTPase activation by the RNA, C86 and C86G reduce GTPase activity by only 50% 734245 3.6.5.4 C87A/C97U site-directed mutagenesis, combining C97U with C87A generates a superactive SRP RNA double mutant that hydrolyzes GTP 5.5fold faster than wild-type SRP RNA 734245 3.6.5.4 C97U site-directed mutagenesis, the mutant prolongs GTPase docking at the distal end, which correlates with its faster GTP hydrolysis rate 734245 3.6.5.4 C97U/G99A site-directed mutagenesis, combining G99A with C87A generate s a superactive SRP RNA double mutant that hydrolyzes GTP 4.6fold faster than wild-type SRP RNA 734245 3.6.5.4 D137A site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview 718911 3.6.5.4 D163A site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview 718911 3.6.5.4 D181N hydrolysis of XTP favored over GTP 644180 3.6.5.4 D181N site-directed mutagenesis of SRP54 (or SRbeta), the mutant shows the wild-type phenotype and binding of SRalpha at wild-type level, but no binding in absence of GTP. SRalpha binding to D181N can be restored to some level by adding back nucleotide, i.e. GTP, GDP, XTP or XDP, but not with ATP 757430 3.6.5.4 D253N no growth defect -, 658538 3.6.5.4 D329A diminished GTPase activity 644174 3.6.5.4 DELTAflhF1 flhF fragment from bp +282 to +1071 relative to the translational initiation site 688862 3.6.5.4 DELTAflhF2 flhF fragment from bp +210 to +685 relative to the translational initiation site 688862 3.6.5.4 E157Q reduced affinity for GTP 644176 3.6.5.4 E475K mutant, complex formation defective 687905 3.6.5.4 F240V mutation in the N-domain modulates the interaction kinetic between Ffh and FtsY 688395 3.6.5.4 F33L mutation in the N-domain modulates the interaction kinetic between cpSRP54 and cpFtsY 688395 3.6.5.4 F48A the cpFtsY mutant exhibits a GTP hydrolysis rate 4times greater than wild type cpFtsY in the absence of liposomes, the mutation reduces light-harvesting chlorophyll-binding protein integration efficiency by nearly 80% 698898 3.6.5.4 F49A the mutation reduces light-harvesting chlorophyll-binding protein integration efficiency by nearly 40% 698898 3.6.5.4 F71V mutation in the N-domain modulates the interaction kinetic between cpSRP54 and cpFtsY 688395 3.6.5.4 G110S reduced GTP hydrolysis, no effect on the interaction with FtsY 658538 3.6.5.4 G118L hydrolysis of XTP favored over GTP 644180 3.6.5.4 G118L site-directed mutagenesis of SRP54 (or SRbeta), the mutant shows a temperature-sensitive phenotype and binding of SRalpha at wild-type level 757430 3.6.5.4 G118L/D181N hydrolysis of XTP favored over GTP 644180 3.6.5.4 G118L/D181N site-directed mutagenesis of SRP54 (or SRbeta) and weak binding of SRalpha 757430 3.6.5.4 G141V substitution introduced into the putative GTP binding motif GXXGXGK 684117 3.6.5.4 G222V substitution introduced into the putative GTP binding motif GXXGXGK 684117 3.6.5.4 G256A no growth defect -, 658538 3.6.5.4 G257A residue resides at the N-GTPase domain interface, mutation produces a lethal phenotype, it does not significantly affect Ffh function, but severely reduces interaction with FtsY -, 658538 3.6.5.4 G455V mutant, defective in SRP-FtsY complex formation 687902 3.6.5.4 G455W mutant, complex formation defective 687905 3.6.5.4 G455W mutant, exhibits no significant GTP hydrolysis and defective in SRP-FtsY complex formation 687902 3.6.5.4 G83A deletion or substitution of G83 by any other nucleotide completely abolishes the stimulatory effect of the SRP RNA on GTP hydrolysis. Despite defective GTP hydrolysis, the G83A mutant shows any detectable defect in the efficiency of GTPase docking at the distal end 734245 3.6.5.4 G90L mutant 644176 3.6.5.4 G99A site-directed mutagenesis, the mutant prolongs GTPase docking at the distal end, which correlates with its faster GTP hydrolysis rate 734245 3.6.5.4 GAK-VSG substitution introduced into the putative GTP binding motif GXXGXGK 684117 3.6.5.4 GTK-VSG substitution introduced into the putative GTP binding motif GXXGXGK 684117 3.6.5.4 H119L hydrolysis of XTP favored over GTP 644180 3.6.5.4 H119L site-directed mutagenesis of SRP54 (or SRbeta), the mutant shows the wild-type phenotype and binding of SRalpha at wild-type level 757430 3.6.5.4 H91L reduced GTPase activity 644176 3.6.5.4 H91L/E157Q mutant 644176 3.6.5.4 H91L/N154A/E157A mutant 644176 3.6.5.4 H91L/N154I mutant 644176 3.6.5.4 K147G substitution introduced into the putative GTP binding motif GXXGXGK 684117 3.6.5.4 K228G substitution introduced into the putative GTP binding motif GXXGXGK 684117 3.6.5.4 K399A mutant, complex formation defective 687905 3.6.5.4 K51A/T52A mutant 644176 3.6.5.4 K51A/T52A/E157Q mutant 644176 3.6.5.4 K51A/T52A/H91L mutant 644176 3.6.5.4 K51I reduced nucleotide affinity 644176 3.6.5.4 K51I/H91L mutant 644176 3.6.5.4 K51I/N154I mutant 644176 3.6.5.4 K751/H119L hydrolysis of XTP favored over GTP 644180 3.6.5.4 K75I hydrolysis of XTP favored over GTP 644180 3.6.5.4 K75I site-directed mutagenesis of SRP54 (or SRbeta), the mutant shows a temperature-sensitive phenotype and no binding of SRalpha 757430 3.6.5.4 K75I/H119L site-directed mutagenesis of SRP54 (or SRbeta), the mutant shows a null mutant phenotype and no binding of SRalpha 757430 3.6.5.4 L164Y the mutant shows no interaction with synthetic L18 peptide 701227 3.6.5.4 L195P reduced GTP hydrolysis, no effect on the interaction with FtsY 658538 3.6.5.4 L199F mutation in the N-domain modulates the interaction kinetic between Ffh and FtsY 688395 3.6.5.4 additional information 4.5S RNA tetraloop mutants UUCG, UUUU, CUUC, GUAA and GAAA 690025 3.6.5.4 additional information binding kinetics and competing inhibitions with several bacterial cpFtsY mutants, affinities of mutant bacterial cpFtsYs for cpSRP54, and complex formations, overview 718911 3.6.5.4 additional information construction of a bi-cistronic construct containing human SRalpha subunit and murine SRbeta subunit lacking the transmembrane domain and cloning in the pET16b vector. Linker deletion variants of SR are created using the same construct 757758 3.6.5.4 additional information construction of several deletion mutants: SRbeta-DELTATM, SRbetaDELTA4, SRbetaDELTA5, SRbeta1C1-DELTATM, SRbeta-DELTAloop, and SRbeta-loop2, phenotypes, altered SRalpha binding of the truncated mutants, overview. Deletion of the last 6 amino-acids from the carboxyl-terminus of the SRbeta GTPase domain results in a molecule (SRbetaC1) with primarily type I topology, similar to SRbeta 757430 3.6.5.4 additional information construction of the FtsYDELTAN1 mutant, and creation of a fusion construct comprising two FtsYDELTAN1 molecules linked via a 31-amino-acid GS linker, similar to the situation for the FtsY/Ffh heterodimer (FtsYDELTAN12) 758489 3.6.5.4 additional information flhF::cat mutant strain assembles flagella and is motile -, 659049 3.6.5.4 additional information FtsY mutated in the 4th GTP-binding consensus element displays reduced GTP-binding and -hydrolysis which correlates with a reduced ability to interact with SRP 644170 3.6.5.4 additional information several mutants show higher GTPase activity than wild-type SRP RNA, most notably mutations at G99, U12, and C97. By modifying the GTPase docking interface, the efficiency of activation of the Ffh-FtsY GTPase complex can be specifically tuned. When G83 is mutated, substitution of C86 with guanine rescues the SRP RNA-mediated stimulation of GTPase activity to 50% of wild-type rate. Despite defective GTP hydrolysis, neither the G83A nor C86G mutant shows any detectable defect in the efficiency of GTPase docking at the distal end 734245 3.6.5.4 additional information SRbeta-deltaloop, hydrolysis of XTP favored over GTP 644180 3.6.5.4 additional information SRbeta-deltaTM, hydrolysis of XTP favored over GTP 644180 3.6.5.4 additional information Srbeta-loop2, hydrolysis of XTP favored over GTP 644180 3.6.5.4 additional information SRbetaC1-deltaTM, hydrolysis of XTP favored over GTP 644180 3.6.5.4 additional information SRbetaD4, hydrolysis of XTP favored over GTP 644180 3.6.5.4 additional information SRbetaD5, hydrolysis of XTP favored over GTP 644180 3.6.5.4 additional information two-site binding analysis of the SRP54DELTAC-ribosome interaction. Having a signal sequence fused to SRP54 or presented by the ribosome does not seem to significantly change SRP binding to the ribosome 757857 3.6.5.4 additional information while CtSRalpha complexed with SRbetaDELTAN can bind ribosomes, both CtSRbetaDELTAN alone and the minimal CtSRalpha138/SRbetaDELTAN complex are unable to bind ribosomes. Full-length CtSRalpha alone readily binds to the ribosomes in a sedimentation assay, as well as to canine ribosomes -, 757758 3.6.5.4 N154A/E157A mutant 644176 3.6.5.4 N154I impaired nucleotide exchange 644176 3.6.5.4 N178K hydrolysis of XTP favored over GTP 644180 3.6.5.4 N178K site-directed mutagenesis of SRP54 (or SRbeta), the mutant shows a temperature-sensitive phenotype and weak binding of SRalpha 757430 3.6.5.4 N302A mutant, GTPase activation defective 687905 3.6.5.4 P142L reduced GTP hydrolysis, no effect on the interaction with FtsY 658538 3.6.5.4 P46A/Q47A/N48A/DELTAS49 mutant 644176 3.6.5.4 Q109A mutant, GTPase activation defective 687905 3.6.5.4 R140A site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview 718911 3.6.5.4 R141A mutant, GTPase activation defective 687905 3.6.5.4 R166A site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview 718911 3.6.5.4 R194A mutant, GTPase activation defective 687905 3.6.5.4 R255N no growth defect -, 658538 3.6.5.4 R333A mutant, GTPase activation defective 687905 3.6.5.4 R386A mutant, GTPase activation defective 687905 3.6.5.4 S220A bypass requirement for GEF 644176 3.6.5.4 S49A reduced GTP hydrolysis 644176 3.6.5.4 S54C/K407C site-directed mutagenesis, mutations S54C in the N-domain and K407C in the M-domain of cpSRP54r resulting in mutant cpSRP54S54C/K407C 756198 3.6.5.4 T226S substitution introduced into the putative GTP binding motif GXXGXGK 684117 3.6.5.4 T307A mutant, complex formation and GTPase activation defective 687905 3.6.5.4 T326N diminished GTPase activity 644174 3.6.5.4 T52N increased affinity for GEF, reduced affinity for GTP 644176 3.6.5.4 T66A prevents GTP-dependent interaction with GAP 644176 3.6.5.4 Thr112Ala deficient in GTP hydrolysis -, 644178 3.6.5.4 V339N/L370N site-directed mutagenesis, mutant cpSRP54V339N/L370N of the mature enyme 756198 3.6.5.4 Y204A the mutant shows no interaction with synthetic L18 peptide 701227