共查询到20条相似文献,搜索用时 31 毫秒
1.
A Bollen R Lathe A Herzog D Denicourt J P Lecocq L Desmarez R Lavallé 《Journal of molecular biology》1979,132(2):219-233
Localized P1 mutagenesis has been used to isolate conditionally lethal mutations in the four-minute region of the Escherichia coli genome. One such mutation, ts25, has been mapped at about 3.7 minutes between the popC and dapD genes. This mutation leads to thermosensitivity of growth and impaired in vivo assembly of 30 S ribosomal subunits at 42 °C. The strain carrying the mutation has an altered S2 ribosomal protein as judged by (1) its inability to maintain stable complex with the ribosome under mild washing conditions and (2) its altered electrophoretic mobility.Spontaneous reversion to temperature independence can restore both the normal assembly in vivo of 30 S ribosomal subunits at 42 °C and the normal electrophoretic behaviour of the S2 ribosomal protein in vitro.We conclude therefore that the ts25 mutation affects the structural gene for ribosomal protein S2 (rpsB). 相似文献
2.
Localized P1 mutagenesis was used to screen for conditionally lethal mutations in ribosomal protein genes. One such mutation, 2859mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and cotransduces at 98% with rpsE (S5). The 2869mis mutation leads to thermosensitivity and impaired assembly in vivo of 50 S ribosomal particles at 42 °C. The strain carrying the mutation has an altered L24 ribosomal protein which at 42 °C shows weaker affinity for 23 S RNA than the wild-type protein. The mutational alteration involves a replacement of glycine by aspartic acid in protein L24 from the mutant. We conclude therefore that the 2859mis mutation affects the structural gene for protein L24 (rplX). 相似文献
3.
Qiang Guo Simon Goto Yuling Chen Boya Feng Yanji Xu Akira Muto Hyouta Himeno Haiteng Deng Jianlin Lei Ning Gao 《Nucleic acids research》2013,41(4):2609-2620
Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3′-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3′-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3′-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences. 相似文献
4.
Albert Herzog Makoto Yaguchi Teresa Cabezón Maria-Clara Corchuelo Jean Petre Alex Bollen 《Molecular & general genetics : MGG》1979,171(1):15-22
Summary The conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ). 相似文献
5.
A thermosensitive mutant (JE386) of Escherichia coli which harbours an alteration in protein S5 of the smaller ribosomal subunit has been isolated. Genetic studies have shown that the lesion causing thermosensitivity also causes the alteration in protein S5, and that this mutation is not in the structural gene for S5 (rpsE). Hence the mutation has been termed rimJ (ribosomal modification). Protein-chemical studies of protein S5 purified from JE386 and its wild-type parent indicated an alteration in the N-terminal tryptic peptide. Amino acid sequence analysis of the N-terminal peptides showed complete homology between wild-type and mutant, suggesting that the N-terminal modification (acetylation) of the parent was absent in the mutant. Gradient transmission mapping has located the rimJ mutation at 31 minutes on the current E. coli genetic map. By constructing a derivative of the mutant heterozygous for rimJ, it has been found that the wild-type allele is dominant over the mutant one. Ts+ revertants of JE386 have been isolated which show either a wild-type ribosomal protein electrophoresis pattern, or an additional alteration in either protein S4 or S5. The mutations in S4 and S5 may compensate the lesion caused by the rimJ mutation of JE386, that is even though the N-terminus of S5 remains unacetylated, bacteria can grow at 42 °C. Furthermore, a mutation near or at strA carried by JE386 has been found to be involved in the phenotypic expression of the rimJ mutation. This mutation was also found to be present in four other strA mutants. Possible implications of the modification of ribosomal proteins in vivo are discussed. 相似文献
6.
Shoji S Dambacher CM Shajani Z Williamson JR Schultz PG 《Journal of molecular biology》2011,413(4):751-761
Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function and may ultimately allow systematic substitution of RPs with RNA. 相似文献
7.
The structural gene for the ribosomal protein S18 in Escherichia coli. I. Genetic studies on a mutant having an alteration in the protein S18 总被引:10,自引:0,他引:10
A Bollen M Faelen J P Lecocq A Herzog J Zengel L Kahan M Nomura 《Journal of molecular biology》1973,76(4):463-472
A mutant of Escherichia coli strain CR341, originally isolated as a temperature-sensitive mutant, was found to have an altered 30 S ribosomal protein (S18) in addition to and independently of temperature sensitivity. Protein S18 from the mutant strain differs in electrophoretic mobility in polyacrylamide gel electrophoresis at pH 4.5 from protein S18 of the parental origin. The mutation responsible for the alteration in S18 is different from two other mutations in the mutant strain which give the temperature-sensitive phenotype. The gene involved in the S18 alteration is located in a region between 76 and 88 minutes on the E. coli genetic map; the location is outside the str-spc region at 64 minutes, where several known ribosomal protein genes are located. An episome covering the loci rha (76 min) through pyr B (84 min) was introduced into the mutant. The resultant merodiploid strains were shown to produce both the normal and the mutant forms of S18. The results support the conclusion described in the accompanying paper (Kahan et al., 1973) that the mutation studied is in the structural gene for S18. 相似文献
8.
The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor. 相似文献
9.
Zhengdong D. Zhang Madhavi Nayar David Ammons Joanne Rampersad George E. Fox 《Journal of microbiological methods》2009,76(2):181-187
A partial knockout compensation method to screen 5S ribosomal RNA sequence variants in vivo is described. The system utilizes an Escherichia coli strain in which five of eight genomic 5S rRNA genes were deleted in conjunction with a plasmid which is compensatory when carrying a functionally active 5S rRNA. The partial knockout strain is transformed with a population of potentially compensatory plasmids each carrying a randomly generated 5S rRNA gene variant. a The ability to compensate the slow growth rate of the knockout strain is used in conjunction with sequencing to rapidly identify variant 5S rRNAs that are functional as well as those that likely are not. The assay is validated by showing that the growth rate of 15 variants separately expressed in the partial knockout strain can be accurately correlated with in vivo assessments of the potential validity of the same variants. A region of 5S rRNA was mutagenized with this approach and nine novel variants were recovered and characterized. Unlike a complete knockout system, the method allows recovery of both deleterious and functional variants.. The method can be used to study variants of any 5S rRNA in the E. coli context including those of E. coli. 相似文献
10.
Nariai M Tanaka T Okada T Shirai C Horigome C Mizuta K 《Nucleic acids research》2005,33(14):4553-4562
Rrs1p, a ribosomal protein L11-binding protein, has an essential role in biogenesis of 60S ribosomal subunits. We obtained conditionally synthetic lethal allele with the rrs1-5 mutation and determined that the mutation is in REX1, which encodes an exonuclease. The highly conserved leucine at 305 was substituted with tryptophan in rex1-1. The rex1-1 allele resulted in 3′-extended 5S rRNA. Polysome analysis revealed that rex1-1 and rrs1-5 caused a synergistic defect in the assembly of 60S ribosomal subunits. In vivo and in vitro binding assays indicate that Rrs1p interacts with the ribosomal protein L5–5S rRNA complex. The rrs1-5 mutation weakens the interaction between Rrs1p with both L5 and L11. These data suggest that the assembly of L5–5S rRNA on 60S ribosomal subunits coordinates with assembly of L11 via Rrs1p. 相似文献
11.
Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and 15N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with 15N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells. 相似文献
12.
Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5′ domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation. 相似文献
13.
Hiroko Nashimoto Akiko Miura Haruo Saito Hisao Uchida 《Molecular & general genetics : MGG》1985,199(3):381-387
Summary Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene. 相似文献
14.
Junichiro Futami Yuki Atago Akari Azuma Endy Widya Putranto Rie Kinoshita Hitoshi Murata Masakiyo Sakaguchi 《Biochemistry and Biophysics Reports》2016
It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems. 相似文献
15.
This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 Å. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry. 相似文献
16.
Alexey P. Korepanov Anna V. Korobeinikova Sergey A. Shestakov Maria B. Garber George M. Gongadze 《Nucleic acids research》2012,40(18):9153-9159
In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained. 相似文献
17.
In vivo stability, maturation and relative differential synthesis rates of individual ribosomal proteins in Escherichia coli B/r 总被引:15,自引:0,他引:15
P P Dennis 《Journal of molecular biology》1974,88(1):25-41
The relative differential synthesis rates2 of individual ribosomal proteins (r-proteins) were determined for Escherichia coli B/r growing in succinate medium (growth rate, μ = 0.65 doublings per hour), glucose medium (μ = 1.36) and glucose-amino acids medium (μ = 1.90). These differential synthesis rates were found to increase co-ordinately with increasing bacterial growth rates; this implies that ribosomes from bacteria growing at different rates are homogeneous with respect to their protein composition (i.e. the stoichiometric amounts of the different r-proteins per ribosome are constant and independent of the bacterial growth rate). Following incorporation into ribosomes, the bulk of the r-proteins were found to be as stable as total protein. Only two r-proteins, S6 and S21, were less stable than total protein; their decay half-lives, measured in succinate and glucose-amino acids cultures, were estimated to be approximately 500 minutes. In addition, post-translational modification of proteins S18, L6 and L11 was observed and the possible relations between modification and in vivo ribosome assembly are discussed. Finally, evidence is presented suggesting that the coordinate production of r-proteins may result, in part, from a mechanism that degrades excess r-proteins that are not rapidly incorporated into ribosomal particles. 相似文献
18.
A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region. 相似文献
19.
In response to DNA damage, Escherichia coli cells activate the expression of the toxin gene tisB of the toxin–antitoxin system tisB-istR1. Of three isoforms, only the processed, highly structured +42 tisB mRNA is active. Translation requires a standby site, composed of two essential elements: a single-stranded region located 100 nucleotides upstream of the sequestered RBS, and a structure near the 5′-end of the active mRNA. Here, we propose that this 5′-structure is an RNA pseudoknot which is required for 30S and protein S1-alone binding to the mRNA. Point mutations that prevent formation of this pseudoknot inhibit formation of translation initiation complexes, impair S1 and 30S binding to the mRNA, and render the tisB mRNA non-toxic in vivo. A set of mutations created in either the left or right arm of stem 2 of the pseudoknot entailed loss of toxicity upon overexpression of the corresponding mRNA variants. Combining the matching right-left arm mutations entirely restored toxicity levels to that of the wild-type, active mRNA. Finally, since many pseudoknots have high affinity for S1, we predicted similar pseudoknots in non-homologous type I toxin–antitoxin systems that exhibit features similar to that of tisB-IstR1, suggesting a shared requirement for standby acting at great distances. 相似文献
20.
Michel De Wilde Teresa Cabezón Raimundo Villarroel Albert Herzog Alex Bollen 《Molecular & general genetics : MGG》1975,142(1):19-33
Summary Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin.Biochemical analysis shows that the mutants each have two modified 30S ribosomal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5). 相似文献