DNase and DMS, other reagents are commonly used to footprint protein–DNA complexes by breaking DNA except where it is protected by bound proteins. For example, organometallic complexes containing copper or iron act by generating hydroxyl radicals that attack and break DNA strands.

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#Unit 3. Fundamental Processes

The charging of a tRNA with its cognate amino acid is a remarkably accurate process.

Experimental measurements indicate, for example, that at equal concentrations of isoleucine and valine, IleRS transfers ∼40,000 isoleucines to tRNAIle for every valine it so transfers.

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#Unit 3. Fundamental Processes

This rejection of noncognate tRNAs at a stage of the reaction that precedes the synthesis of misacylated tRNA is sometimes referred to as kinetic proofreading.

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#Unit 3. Fundamental Processes

 Valyl-tRNA synthetase can sterically exclude isoleucine from its catalytic pocket because isoleucine is larger than valine. In contrast, valine should slip easily into the catalytic pocket of the isoleucyl-tRNA synthetase. Although both amino acids will fit into the isoleucyl-tRNA synthetase amino acid–binding site, interactions with the extra bonding with tyrosine’s OH group. Since no other amino acid resembles tyrosine, the enzyme can do without an editing function

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#Unit 3. Fundamental Processes

isoleucyl-tRNA synthetase has a nearby editing pocket (a deep cleft in the enzyme) that allows it to proofread the product of the adenylylation reaction. AMP-valine (as well as adenylylates of other small amino acids, such as alanine) can fit into this editing pocket, where it is hydrolyzed and released as free valine and AMP. In contrast, AMP-isoleucine is too large to enter the editing pocket and is therefore not subject to hydrolysis. As a consequence, isoleucyl-tRNA synthetase discriminates against valine twice: in the initial binding and adenylylation of the amino acid (discriminating by a factor of 100), and then in the editing of the adenylylated amino acid (again discriminating by a factor of 100), for an

overall selectivity of 10,000-fold (i.e., an error rate of 0.01%).

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#Unit 3. Fundamental Processes

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#Unit 3. Fundamental Processes

ThrRS has the opposite problem: It must synthesize Thr–tRNAThr but not Val–tRNAThr. Specificity is conferred by the aminoacylation site, which contains a Zn2+ ion that is coordinated by the side chain OH group of threonine. Valine cannot coordinate the Zn2+ in this way and hence does not undergo adenylylation by ThrRS.  A separate editing site deals with misacylated Ser–tRNAThr. TyrRS distinguishes between tyrosine and phenylalanine through hydrogen missing from the repertoire are GlnRS and asparaginyl-tRNA synthetase (AsnRS). To synthesize Gln-tRNAGln and Asn-tRNAAsn, these organisms possess distinct glutamyl-tRNA synthetase (GluRS) and aspartyl-tRNA synthetase (AspRS) enzymes that are nondiscriminating (ND). GluRSND synthesizes both Glu-tRNAGlu as well as misacylated Glu-tRNAGln; AspRSND synthesizes both Asp-tRNAAsp and misacylated Asp-tRNAAsn.