Showing papers by "Robert T. Sauer published in 2002"

Stop codons preceded by rare arginine codons are efficient determinants of SsrA tagging in Escherichia coli

Abstract: The SsrA or tmRNA quality control system intervenes when ribosomes stall on mRNAs and directs the addition of a C-terminal peptide tag that targets the modified polypeptide for degradation. Although hundreds of SsrA-tagged proteins can be detected in cells when degradation is prevented, most of these species have not been identified. Consequently, the mRNA sequence determinants that cause ribosome stalling and SsrA tagging are poorly understood. SsrA tagging of Escherichia coli ribokinase occurs at three specific sites at or near the C terminus of this protein. The sites of tagging correspond to ribosome stalling at the termination codon and at rare AGG codons encoding Arg-307 and Arg-309, the antepenultimate and C-terminal residues of E. coli ribokinase. Mutational analyses and studies of the effects of overexpressing the tRNA that decodes AGG reveal that the combination of a rare arginine codon at the C terminus and the adjacent inefficient UGA termination codon act to recruit the SsrA-tagging system, presumably by slowing the rate of translation elongation and termination.

Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects.

Abstract: Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, α-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including λ and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 ± 0.9 cal mol−1 site−1. However, β-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.