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DnaA protein/DNA interaction. Modulation of the recognition sequence
1Max-Planck-Institut für molekulare Genetik, Abt. Trautner, Berlin, Federal Republic of Germany.
Abstract:
In the active orientation the DnaA protein/dnaA box complex blocks transcribing RNA polymerase. The extent of transcription termination at different dnaA boxes was used to determine in vivo their various affinities to the DnaA protein. The rate of transcription distal to the respective dnaA box was monitored by the expression of the reporter gene galK. The dnaA boxes (5'-TTATACACA and 5'-TTATCCAAA), present in oriC, showed the strongest binding affinity. The dnaA box 5'-TTTTCCACA was mutated at eight positions such that the boxes differed from the consensus sequence, 5'-TT(A/T)T(A/C)CA(A/C)A, defined so far. Based on the different properties of the dnaA boxes, a new consensus sequence was derived: 5'-(T/C)(T/C)(A/T/C)T(A/C)C(A/G)(A/C/T(A/C).
Insights
Researchers studied DNA replication initiation by analyzing DnaA protein binding to dnaA boxes. They found specific dnaA box sequences in oriC have the highest affinity, leading to a refined consensus sequence for DnaA protein binding.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The DnaA protein is crucial for initiating DNA replication in bacteria.
- DnaA protein binds to specific DNA sequences called dnaA boxes, primarily within the origin of replication (oriC).
- The binding affinity of DnaA to dnaA boxes influences the regulation of DNA replication initiation.
Purpose of the Study:
- To investigate the in vivo binding affinities of various dnaA boxes to the DnaA protein.
- To determine the functional significance of different dnaA box sequences in regulating transcription termination.
- To derive a new, more accurate consensus sequence for dnaA boxes based on their binding properties.
Main Methods:
- Assessing DnaA protein/dnaA box complex interactions by monitoring transcription termination.
- Quantifying DnaA protein binding affinities in vivo using different dnaA box sequences.
- Measuring the rate of transcription distal to dnaA boxes via reporter gene (galK) expression.
- Mutating a known dnaA box sequence at eight positions to create variants with altered properties.
Main Results:
- The dnaA boxes 5'-TTATACACA and 5'-TTATCCAAA, located in oriC, exhibited the strongest binding affinity for the DnaA protein.
- The study identified varying affinities of different dnaA boxes to the DnaA protein.
- Mutational analysis of the 5'-TTTTCCACA dnaA box provided insights into sequence-dependent binding.
Conclusions:
- The binding affinity of dnaA boxes to the DnaA protein is sequence-dependent and can be quantified in vivo.
- Specific dnaA box sequences within oriC are critical for high-affinity DnaA binding.
- A refined consensus sequence, 5'-(T/C)(T/C)(A/T/C)T(A/C)C(A/G)(A/C/T(A/C), better represents the functional dnaA box sequences crucial for DNA replication initiation.