Simultaneous Binding of Multiple EF-Tu Copies to Translating Ribosomes in Live Escherichia coli

ABSTRACT In bacteria, elongation factor Tu is a translational cofactor that forms ternary complexes with aminoacyl-tRNA (aa-tRNA) and GTP. Binding of a ternary complex to one of four flexible L7/L12 units on the ribosome tethers a charged tRNA in close proximity to the ribosomal A site. Two sequential tests for a match between the aa-tRNA anticodon and the current mRNA codon then follow. Because one elongation cycle can occur in as little as 50 ms and the vast majority of aa-tRNA copies are not cognate with the current mRNA codon, this testing must occur rapidly. We present a single-molecule localization and tracking study of fluorescently labeled EF-Tu in live Escherichia coli. Imaging at 2 ms/frame distinguishes 60% slowly diffusing EF-Tu copies (assigned as transiently bound to translating ribosome) from 40% rapidly diffusing copies (assigned as a mixture of free ternary complexes and free EF-Tu). Combining these percentages with copy number estimates, we infer that the four L7/L12 sites are essentially saturated with ternary complexes in vivo. The results corroborate an earlier inference that all four sites can simultaneously tether ternary complexes near the A site, creating a high local concentration that may greatly enhance the rate of testing of aa-tRNAs. Our data and a combinatorial argument both suggest that the initial recognition test for a codon-anticodon match occurs in less than 1 to 2 ms per aa-tRNA copy. The results refute a recent study (A. Plochowietz, I. Farrell, Z. Smilansky, B. S. Cooperman, and A. N. Kapanidis, Nucleic Acids Res 45:926–937, 2016, https://doi.org/10.1093/nar/gkw787) of tRNA diffusion in E. coli that inferred that aa-tRNAs arrive at the ribosomal A site as bare monomers, not as ternary complexes. IMPORTANCE Ribosomes catalyze translation of the mRNA codon sequence into the corresponding sequence of amino acids within the nascent polypeptide chain. Polypeptide elongation can be as fast as 50 ms per added amino acid. Each amino acid arrives at the ribosome as a ternary complex comprising an aminoacyl-tRNA (aa-tRNA), an elongation factor called EF-Tu, and GTP. There are 43 different aa-tRNAs in use, only one of which typically matches the current mRNA codon. Thus, ternary complexes must be tested very rapidly. Here we use fluorescence-based single-molecule methods that locate and track single EF-Tu copies in E. coli. Fast and slow diffusive behavior determines the fraction of EF-Tu copies that are ribosome bound. We infer simultaneous tethering of ~4 ternary complexes to the ribosome, which may facilitate rapid initial testing for codon matching on a time scale of less than 1 to 2 ms per aa-tRNA.

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

Biochemical Journal ◽

10.1042/bj2960851 ◽

1993 ◽

Vol 296 (3) ◽

pp. 851-857 ◽

Cited By ~ 23

Author(s):

T Belyaeva ◽

L Griffiths ◽

S Minchin ◽

J Cole ◽

S Busby

Keyword(s):

Escherichia Coli ◽

Point Mutations ◽

Growth Conditions ◽

Upstream Region ◽

E Coli ◽

Run Off ◽

A Site ◽

Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

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Genetic and Functional Analyses of the Conserved C-Terminal Core Domain of Escherichia coli FtsZ

Journal of Bacteriology ◽

10.1128/jb.181.24.7531-7544.1999 ◽

1999 ◽

Vol 181 (24) ◽

pp. 7531-7544 ◽

Cited By ~ 175

Author(s):

Xiaolan Ma ◽

William Margolin

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Immunoblot Analysis ◽

Conserved Residues ◽

Core Domain ◽

Mutant Proteins ◽

E Coli ◽

Stable Mutant ◽

Functional Analyses

ABSTRACT In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. coliFtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomalftsZ mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.

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The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-CoutConfiguration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

Journal of Bacteriology ◽

10.1128/jb.00423-16 ◽

2016 ◽

Vol 198 (23) ◽

pp. 3186-3199 ◽

Cited By ~ 4

Author(s):

Amit Pathania ◽

Arvind Kumar Gupta ◽

Swati Dubey ◽

Balasubramanian Gopal ◽

Abhijit A. Sardesai

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Cytoplasmic Membrane ◽

Basic Amino Acid ◽

Intramolecular Interactions ◽

Membrane Topology ◽

Content Type ◽

E Coli ◽

Terminal Domain

ABSTRACTArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export ofl-arginine (Arg) inEscherichia coliandl-lysine (Lys) and Arg inCorynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane ofE. coli, we have employed a combination of cysteine accessibilityin situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Coutconfiguration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO functionin vivoand that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO functionin vivoand their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomerin vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.IMPORTANCEThe orthologous proteins LysE ofC. glutamicumand ArgO ofE. colifunction as exporters of the basic amino acidsl-arginine andl-lysine and the basic amino acidl-arginine, respectively, and LysE can functionally substitute for ArgO when expressed inE. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic andin silicostudies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

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Protein Synthesis in Escherichia coli with Mischarged tRNA

Journal of Bacteriology ◽

10.1128/jb.185.12.3524-3526.2003 ◽

2003 ◽

Vol 185 (12) ◽

pp. 3524-3526 ◽

Cited By ~ 29

Author(s):

Bokkee Min ◽

Makoto Kitabatake ◽

Carla Polycarpo ◽

Joanne Pelaschier ◽

Gregory Raczniak ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Synthesis ◽

Elongation Factor ◽

Trna Synthetase ◽

Wild Type ◽

Wild Type Level ◽

E Coli ◽

Missense Mutant ◽

Tryptophan Synthetase

ABSTRACT Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNAAsp, while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNAAsn, a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNAAsn and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNAAsn are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.

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The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation

Journal of Biological Chemistry ◽

10.1074/jbc.m607058200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32303-32309 ◽

Cited By ~ 53

Author(s):

Taeko Komoda ◽

Neuza S. Sato ◽

Steven S. Phelps ◽

Naoki Namba ◽

Simpson Joseph ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Functional Role ◽

Elongation Factor ◽

Normal Growth ◽

Wild Type ◽

Reading Frame ◽

E Coli ◽

Translational Activity ◽

A Site

Helix 38 (H38) in 23 S rRNA, which is known as the “A-site finger (ASF),” is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

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Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells

Nature Communications ◽

10.1038/ncomms11641 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 25

Author(s):

Burak Okumus ◽

Dirk Landgraf ◽

Ghee Chuan Lai ◽

Somenath Bakshi ◽

Juan Carlos Arias-Castro ◽

...

Keyword(s):

Escherichia Coli ◽

Single Molecule ◽

Exponential Phase ◽

Regulatory Proteins ◽

Adapter Protein ◽

Average Cell ◽

E Coli ◽

Slowing Down ◽

Automated Microscopy

AbstractMany key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of liveEscherichia coli(E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of theE. colistress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3–4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting.

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In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli

10.22541/au.161417238.80994704/v1 ◽

2021 ◽

Author(s):

Filipe Silva ◽

Sara Santos ◽

Roberto Meyer ◽

Eduardo Silva ◽

Carina Pinheiro ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Target Protein ◽

E Coli ◽

Recombinant Protein Purification ◽

Intracellular Processing ◽

Specific Protease ◽

A Site

Recombinant proteins are generally fused with solubility enhancer tags to improve target protein folding and solubility. However, the fusion protein strategy usually requires the use of expensive proteases to perform in vitro proteolysis and additional chromatography steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, are useful for simplifying the recombinant protein purification process, for screening molecules that fail to remain soluble after tag removal, and to promote higher yields of soluble target protein. Herein, we review controlled intracellular processing (CIP) systems, tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular protein processing regarding system design features, significant advantages and limitations of the various strategies.

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Mu-Like Prophage Strong Gyrase Site Sequences: Analysis of Properties Required for Promoting Efficient Mu DNA Replication

Journal of Bacteriology ◽

10.1128/jb.186.14.4575-4584.2004 ◽

2004 ◽

Vol 186 (14) ◽

pp. 4575-4584 ◽

Cited By ~ 3

Author(s):

Mark Oram ◽

Martin L. Pato

Keyword(s):

Escherichia Coli ◽

Dna Gyrase ◽

Bacteriophage Mu ◽

Reporter Plasmid ◽

E Coli ◽

Host Lysis ◽

Sequences Analysis ◽

A Site

ABSTRACT The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.

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