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      Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of “shifty” four-base codons with a library approach in Escherichia coli1

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          Abstract

          Naturally occurring tRNA mutants are known that suppress +1 frameshift mutations by means of an extended anticodon loop, and a few have been used in protein mutagenesis. In an effort to expand the number of possible ways to uniquely and efficiently encode unnatural amino acids, we have devised a general strategy to select tRNAs with the ability to suppress four-base codons from a library of tRNAs with randomized 8 or 9 nt anticodon loops. Our selectants included both known and novel suppressible four-base codons and resulted in a set of very efficient, non-cross-reactive tRNA/four-base codon pairs for AGGA, UAGA, CCCU and CUAG. The most efficient four-base codon suppressors had Watson-Crick complementary anticodons, and the sequences of the anticodon loops outside of the anticodons varied with the anticodon. Additionally, four-base codon reporter libraries were used to identify “shifty” sites at which +1 frameshifting is most favorable in the absence of suppressor tRNAs in Escherichia coli. We intend to use these tRNAs to explore the limits of unnatural polypeptide biosynthesis, both in vitro and eventually in vivo. In addition, this selection strategy is being extended to identify novel five- and six-base codon suppressors.

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          High efficiency transformation of Escherichia coli with plasmids.

          We have re-evaluated the conditions for preparing competent Escherichia coli cells and established a simple and efficient method (SEM) for plasmid transfection. Cells (DH5, JM109 and HB101) prepared by SEM are extremely competent for transformation (1-3 x 10(9) cfu/microgram of pBR322 DNA), and can be stored in liquid nitrogen for at least 40 days without loss of competence. Unlike electroporation, transformation using these competent cells is affected minimally by salts in DNA preparation. These competent cells are particularly useful for construction of high-complexity cDNA libraries with a minimum expenditure of mRNA.
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            Programmed translational frameshifting.

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              Translational accuracy and the fitness of bacteria.

              There are two aspects of the relationship between translational accuracy and the fitness of bacteria that I hope have been clarified in this review. One is that the impact of translational errors on the fitness of bacteria depends very much on nutritional conditions. It would seem that bacterial populations have the capacity to respond to different growth opportunities by the selection of suitable variants. It is particularly surprising how few mutations seem to be required to transform a slowly growing natural isolate with inefficient as well as inaccurate ribosomes into a growth-optimized laboratory strain. It would not be suprising if the selection of the slow, natural isolate phenotype under starvation conditions is equally facile. Another aspect of the accuracy-fitness relationship worth emphasizing is the strong impact of processivity errors and the weak impact of missense errors on the structures of proteins as well as on the growth of cells. What has been learned about translation mechanisms up to now is really only a preliminary to what remains to be discovered about the movements of tRNA, mRNA, and ribosomal subunits that support the processivity of translation. It would be very useful to have more direct methods at hand with which to study these movements. Likewise, the availability of methods to measure processivity errors in natural isolates would help to round out our view of the variability of the ribosomal mechanisms in nature.
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                Author and article information

                Contributors
                Journal
                J Mol Biol
                J. Mol. Biol
                Journal of Molecular Biology
                Academic Press.
                0022-2836
                1089-8638
                25 May 2002
                30 March 2001
                25 May 2002
                : 307
                : 3
                : 755-769
                Affiliations
                [1 ]Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
                [2 ]Department of Chemistry, University of California, Berkeley, CA 94720, USA
                Author notes
                [* ]Corresponding author schultz@ 123456scripps.edu
                [2]

                Present address: T. J. Magliery, Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

                [3]

                T.J.M. and J.C.A. contributed equally to this work.

                Article
                S0022-2836(01)94518-8
                10.1006/jmbi.2001.4518
                7125544
                11273699
                41cc170f-9cba-4ad1-80e8-1033590b60b2
                Copyright © 2001 Academic Press. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 6 November 2000
                : 12 January 2001
                : 19 January 2001
                Categories
                Article

                Molecular biology
                four-base codon,suppressors,genetic code,shifty codons
                Molecular biology
                four-base codon, suppressors, genetic code, shifty codons

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