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The minimal sequence needed to define a functional DNA terminator in Bacillus subtilis
M T Smith1, D B Langley, P A Young
1Department of Biochemistry, University of Sydney NSW, Australia.
Abstract:
The 47 bp DNA replication terminator (IRI) of Bacillus subtilis, contains two binding sites, A and B, for the replication terminator protein (RTP). Each site binds a dimer of RTP. Removal of the first two base-pairs (bp 1-2) from IRI completely destroyed in vivo terminator (fork arrest) function and was accompanied by loss of RTP binding to the A site, which is distal to the approaching fork that is arrested. Removal of base-pairs 34 to 47 from the other end, proximal to the approaching fork, lowered in vivo function to approximately 50% of the complete IRI. RTP binding appeared to be largely unaffected. Terminator function remained at the approximately 50% level with further deletions that proceeded as far as to include base-pair 28; and RTP binding remained largely unaffected. Removal of more of the sequence beyond base-pair 27 and into the region that makes extensive contact with RTP resulted in a further impairment to in vivo function, and caused altered RTP binding. The base-pairs 1 to 24 segment retained only 16% fork arrest activity and the effect on RTP binding was largely evidenced by an elimination of the ability of this extensively truncated sequence to fill the B site alone. The behaviour of the various terminator deletions emphasize the importance of the previously defined RTP-DNA contacts which allow the binding of RTP to the two overlapping sites, A and B, of IRI for terminator function. A comparison of the affinities of selected truncated terminators for RTP raises the possibility that the overall affinity of RTP for its DNA terminator is not the sole determinant of terminator function.
Insights
DNA replication termination in Bacillus subtilis requires specific sequences for replication terminator protein (RTP) binding. RTP binding to both sites A and B on the IRI terminator is crucial for full fork arrest activity.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Bacillus subtilis utilizes a 47 bp DNA replication terminator (IRI) essential for regulating DNA replication termination.
- The IRI terminator contains two binding sites, A and B, for the replication terminator protein (RTP), with each site accommodating an RTP dimer.
Purpose of the Study:
- To investigate the functional significance of specific base pairs within the Bacillus subtilis IRI DNA replication terminator.
- To elucidate the relationship between replication terminator protein (RTP) binding affinity and in vivo terminator function.
Main Methods:
- Systematic deletion mutagenesis of the IRI terminator sequence.
- In vivo assessment of DNA replication fork arrest activity.
- Electrophoretic mobility shift assays (EMSAs) to evaluate RTP binding affinity to truncated IRI sequences.
Main Results:
- Deletion of base pairs 1-2 from IRI abolished in vivo terminator function and RTP binding to site A.
- Deletions at the proximal end (base pairs 34-47) reduced function by ~50% with largely unaffected RTP binding.
- Further deletions into the RTP contact region impaired both in vivo function and RTP binding, with a truncated sequence (bp 1-24) showing minimal activity and altered B site binding.
Conclusions:
- Specific RTP-DNA contacts within the IRI terminator, particularly involving sites A and B, are critical for efficient DNA replication termination.
- While RTP binding is necessary, the overall binding affinity alone may not solely determine the efficacy of DNA replication termination.