Publications (3)12.78 Total impact
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Chapter: The Analysis of Translational Activity Using a Reporter Gene Constructed from Repeats of an Antibody-Binding Domain from Protein A
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ABSTRACT: We describe here the application of the protein A reporter gene 3A′ for in vivo translation studies in Escherichia coli (1–3). The 3A′ gene product is composed of three repeats of the A′ domain, an engineered derivative of the antibody-binding B domain of protein A from Staphylococcus aureus (Fig. 1) (4,5). The sequence of the 3A′ gene is shown in Fig. 2. A polylinker containing unique restriction sites between the regions coding for the second and third A′ domain is used for cloning of test codon sequences (Figs. 1 and 2). The genetic construction steps resulting in the 3A′ gene plasmid in current use are described in Note 15.02/2008: pages 75-91; -
Article: Functional effects of deleting the coiled-coil motif in Escherichia coli elongation factor Ts.
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ABSTRACT: Elongation factor Ts (EF-Ts) is the guanine nucleotide-exchange factor for elongation factor Tu (EF-Tu) that is responsible for promoting the binding of aminoacyl-tRNA to the mRNA-programmed ribosome. The structure of the Escherichia coli EF-Tu-EF-Ts complex reveals a protruding antiparallel coiled-coil motif in EF-Ts, which is responsible for the dimerization of EF-Ts in the crystal. In this study, the sequence encoding the coiled-coil motif in EF-Ts was deleted from the genome in Escherichia coli by gene replacement. The growth rate of the resulting mutant strain was 70-95% of that of the wild-type strain, depending on the growth conditions used. The mutant strain sensed amino acid starvation and synthesized the nucleotides guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate at a lower cell density than the wild-type strain. Deletion of the coiled-coil motif only partially reduced the ability of EF-Ts to stimulate the guanine nucleotide exchange in EF-Tu. However, the concentration of guanine nucleotides (GDP and GTP) required to dissociate the mutant EF-Tu-EF-Ts complex was at least two orders of magnitude lower than that for the wild-type complex. The results show that the coiled-coil motif plays a significant role in the ability of EF-Ts to compete with guanine nucleotides for the binding to EF-Tu. The present results also indicate that the deletion alters the competition between EF-Ts and kirromycin for the binding to EF-Tu.European Journal of Biochemistry 12/2003; 270(21):4294-305. · 3.58 Impact Factor -
Article: Cis control of gene expression in E.coli by ribosome queuing at an inefficient translational stop signal.
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ABSTRACT: An UGA stop codon context which is inefficient because of the 3'-flanking context and the last two amino acids in the gene protein product has a negative effect on gene expression, as shown using a model protein A' gene. This is particularly true at low mRNA levels, corresponding to a high intracellular ribosome/mRNA ratio. The negative effect is smaller if this ratio is decreased, or if the distance between the initiation and termination signals is increased. The results suggest that an inefficient termination codon can cause ribosomal pausing and queuing along the upstream mRNA region, thus blocking translation initiation of short genes. This cis control effect is dependent on the stop codon context, including the C-terminal amino acids in the gene product, the translation initiation signal strength, the ribosome/mRNA ratio and the size of the mRNA coding region. A large proportion of poorly expressed natural Escherichia coli genes are small, and the weak termination codon UGA is under-represented in small, highly expressed E.coli genes as compared with the efficient stop codon UAA.The EMBO Journal 09/2002; 21(16):4357-67. · 9.20 Impact Factor
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2008
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Stockholm University
Stockholm, Stockholm, Sweden
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