For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis-or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when crosslinked show the largest "photoswitch effect," i.e., difference in activity when illuminated with UV vs. blue light, >30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in V max rather than K m .azobenzene | DNA cleavage | endonuclease | photoswitch | PvuII P roteins exist in nature whose activity can be controlled by light; perhaps one of the best known examples is rhodopsin, which is regulated by the cis∕trans isomerization of its cofactor retinal. For many biological applications it would be desirable to selectively switch the activity of a protein on and off by light in a similar manner (1). This could be accomplished by the introduction of a photosensitive compound into the protein of interest. Recent developments in photosensitive compounds such as the azobenzene derivatives have made the scenario a reality. Azobenzene can be reversibly isomerized between the extended trans-and the more compact cis-configuration by illumination with UV (trans → cis) or blue-light (cis → trans) as well as by thermal relaxation (cis → trans) (2-4). Four generally applicable approaches have been used to introduce azobenzene groups into peptides or proteins: (i) incorporation during peptide synthesis (5-8), (ii) incorporation during in vitro translation (9, 10), (iii) incorporation in vivo by using an orthogonal tRNA/aminoacyl tRNA synthetase pair specific for phenylalanine-4′-azobenzene (11), and (iv) chemical modification of peptides and proteins (3,12,13). Another more specific approach is to use azobenzenemodified ligands (e.g., inhibitors) for proteins (14,15). Chemical modification, the most widely used of these approaches, can be done with mono-or bifunctional azobenzene derivatives. Modification with monofunctional azobenzene derivatives relies on steric effects (e.g., interference with ligand binding), whereas modification with bifunctional azobenzene derivat...

(2011) In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillussubtilis, RNA Biology, 8:5,[839][840][841][842][843][844][845][846][847][848][849]

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All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.

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