E. coli cells that are exposed to low doses of an alkylating agent (such as NTG) are better able to handle subsequent high doses of an alkylating agent. In other words, the cells seem to adapt to be able to handle the agent. This is called the adaptive response.
The low initial dose of mutagen induces the synthesis of four gene products (Ada, AlkA, AlkB, AidB) which are then able to deal with the subsequent high dosage.
- Ada is both a methyl
transferase and a transcriptional activator. Its function is
described below.
- AlkA is a glycosylase
that removes N7-methylguanine, N3-methylpurines
and O2-methylpyrimidines. The ABASIC sites generated are
then excised by an AP endonuclease and repaired by PolI and DNA ligase.
- AlkB has an unknown function
in repair. alkB mutants are hypersensitive to
MMS and DMS and are poor at reactivating MMS treated bacteriophage
l.
- AidB resembles the isovaleryl CoA dehydrogenase and may be involved in metabolizing alkylating agents such as NTG.
Ada
The key molecule in the adaptive response is the Ada protein. It is both a repair enzyme and a regulatory protein. The repair activity stimulates the regulatory function; the regulatory action stimulates furthrt repair.
The Ada protein is a methyltransferase which is expressed at low levels in normal cells. Methylation is irreversible.
Its function is to sense the presence of alkylated DNA. If it encounters O-6-methylguanine or O-4-methylthymine, both of which are highly mutagenic, it catalyzes the transfer of the methyl group to its own Cys321. If it encounters a methylated phosphate group, it transfers the methyl group to its own Cys69. While methylated phosphates are innocuous, they serve as an indicator of abnormal methylation activity which is then conveyed to Ada.
Ada methylated at Cys69 becomes a transcriptional activator which functions at three promoters:
- the aidB promoter
transcription from this promoter allows expression of the AidB protein.
- the ada promoter
transcription from this promoter expresses both the ada and alkB genes. Since Ada regulates its own expression, it is autoregulatory.
- the alkA promoter
transcription from this promoter allows expression of AlkA .
Activation at the ada and aidB promoters is mechanistically similar.
RNA polymerase binds poorly to the ada and aidB promoters in the absence of Ada. In both cases, the carboxyl terminal domain (CTD) of the RNA polymerase a subunit (a-CTD) contacts the UP promoter element (an A/T rich region located between 40 and 60 bp upstream of the startpoint of transcription). However, binding of MeAda is required to formation a ternary complex that is capable of transcription initiation.
The amino-terminal domain (NTD) of MeAda binds to its binding site which is located in the UP promoter element (between -57 and -45) in the ada promoter and (between -55 and -43) in the aidB promoter. Binding is independent of RNA polymerase.
The carboxyl terminal domain of MeAda then interacts with the s subunit of RNA polymerase. This interaction is mediated by a region of negatively charged residues (E574, E575, E591, E605) in the s70 subunit of RNA polymerase. This stabilises the otherwise weak interaction between RNA polymerase and the ada promoter. (Note that E575 and E591 have also been implicated in activation by the PhoB and lcI proteins.)
Some evidence:
- The Ada NTD
will bind to DNA by itself as long as Cys69 is methylated. The
affinity and specificity of binding are the same as that of the
full-length protein.
- The Ada NTD
cannot activate transcription at either the ada
or aidB promoters.
It is believed that methylation at Cys69 converts Ada from an inactive to an active regulatory form as a result of a conformational change. Cys321 is buried in the interior of the protein. However, upon methylation of Cys69, the positively charged region around Cys321 becomes exposed on the surface of the protein. Methylation of Cys321 stabilizes the altered conformation that permits interaction of Ada and s70. Methylation of Cys321 per se plays only a small role in activation. While it is not necessary for activation, methylation will result in optimum activation.
Activation at the alkA
promoter is mechanistically different.
At this promoter, the Ada binding site is located between -47 and -35. This positions Ada so that it can interact both with the a-CTD of RNA polymerase and with the s70 subunit.
In this case it is the amino-terminal domain (NTD) of MeAda which is responsible for interacting with RNA polymerase as well as for binding to DNA. The interaction is mediated by positively charged residues (K593, K597, R603) in the s70 subunit of RNA polymerase and (presumably) a negatively charged residue in Ada. There is also an interaction between Ada and the a-CTD of RNA polymerase which contributes to the binding of RNA polymerase to the promoter.
(Note that residues in the 593-603 region of s have been implicated in CRP acivation at some promoters; and R596 is a target for lcI.)
Non-methylated Ada is able to activate the alkA promoter weakly. This does not happen at the ada and aidB promoters.
Ada also plays a role in protecting the cell from methylation during stationary phase. MeAda is also able to interact with the sS subunit of RNA polymerase thereby activating stationary phase specific gene expression.
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Read a more detailed review of the adaptive response: Regulatory Responses of the Adaptive
Response to Alkylation Damage: a Simple Regulon with Complex
Regulatory Features |
