A comparison of the rates of RNA and protein synthesis in Escherichia coli cells partially starved for different amino acids

The effects of oxytetracycline on sensitive and resistant strains of Escherichia coli were studied in relation to: (1) growth of bacteria in shaken cultures, (2) incorporation of amino acids into polypeptides by cell-free extracts, (3) binding of aminoacyl-tRNA to ribosomes. Our results support the hypothesis that resistance to the antibiotic is due to impermeability of bacteria to the drug.Evidence is also presented that there is no irreversible binding of oxytetracycline at ribosomal sites involved in protein synthesis.

Download Full-text

Control of RNA and protein synthesis by the concentration of Trp-tRNATrp in Escherichia coli infected with bacteriophage MS2

Journal of Molecular Biology ◽

10.1016/s0022-2836(83)80073-4 ◽

1983 ◽

Vol 168 (4) ◽

pp. 747-763 ◽

Cited By ~ 5

Author(s):

Stephen W. Koontz ◽

Hieronim Jakubowski ◽

Emanuel Goldman

Keyword(s):

Escherichia Coli ◽

Protein Synthesis ◽

Bacteriophage Ms2 ◽

Rna And Protein Synthesis

Download Full-text

Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms20030492 ◽

2019 ◽

Vol 20 (3) ◽

pp. 492 ◽

Cited By ~ 8

Author(s):

Jiro Adachi ◽

Kazushige Katsura ◽

Eiko Seki ◽

Chie Takemoto ◽

Mikako Shirouzu ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Protein Synthesis ◽

Trna Synthetase ◽

Translation Efficiency ◽

Free System ◽

Free Protein ◽

Cell Free System ◽

Natural Amino Acids ◽

Cell Free Protein Synthesis

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

Download Full-text

Association of nascent polypeptide with 30S ribosomal subunits

Biochemical Journal ◽

10.1042/bj1360859 ◽

1973 ◽

Vol 136 (4) ◽

pp. 859-863 ◽

Cited By ~ 2

Author(s):

Michael Cannon ◽

M. Amin A. Mirza ◽

Margaret L. M. Anderson

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Protein Synthesis ◽

Trichloroacetic Acid ◽

Sucrose Gradient ◽

Gradient Centrifugation ◽

Ribosomal Subunits ◽

Nascent Polypeptide ◽

Sucrose Gradient Centrifugation ◽

Crude Extracts

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg2+concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).

Download Full-text

Extending the cross-linking/mass spectrometry strategy: Facile incorporation of photo-activatable amino acids into the model protein calmodulin in Escherichia coli cells

Methods ◽

10.1016/j.ymeth.2015.02.012 ◽

2015 ◽

Vol 89 ◽

pp. 121-127 ◽

Cited By ~ 18

Author(s):

Christine Piotrowski ◽

Christian H. Ihling ◽

Andrea Sinz

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Amino Acids ◽

Cross Linking ◽

Escherichia Coli Cells ◽

Model Protein ◽

The Cross

Download Full-text

Directed evolution of rRNA improves translation kinetics and recombinant protein yield

Nature Communications ◽

10.1038/s41467-021-25852-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Fan Liu ◽

Siniša Bratulić ◽

Alan Costello ◽

Teemu P. Miettinen ◽

Ahmed H. Badran

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Protein Synthesis ◽

Bacterial Population ◽

Protein Yield ◽

Rrna Sequence ◽

Evolutionary Trajectory ◽

Noncanonical Amino Acids ◽

Rate Limiting

AbstractIn bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Enhanced ribosome kinetics may augment bacterial growth and biomanufacturing through improvements to overall protein yield, but whether this can be achieved by ribosome-specific modifications remains unknown. Here, we evolve 16S ribosomal RNAs (rRNAs) from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae towards enhanced protein synthesis rates. We find that rRNA sequence origin significantly impacted evolutionary trajectory and generated rRNA mutants with augmented protein synthesis rates in both natural and engineered contexts, including the incorporation of noncanonical amino acids. Moreover, discovered consensus mutations can be ported onto phylogenetically divergent rRNAs, imparting improved translational activities. Finally, we show that increased translation rates in vivo coincide with only moderately reduced translational fidelity, but do not enhance bacterial population growth. Together, these findings provide a versatile platform for development of unnatural ribosomal functions in vivo.

Download Full-text