Anyway, it looks like I will finially be learning about bacterial transcription and replication in greater detail, something that needed to happen sooner or later.
DNA replication initiation: mechanisms and regulation in bacteria
Mott, et al. Nature Reviews Microbio 2007
So, bacterial DNA replication initiates at oriC. This sequence is preceded by a lot of special sequences that bind various factors. Many of these are called DnaA boxes, because they help bind DnaA, the initiator protein. This protein oligomerizes in its ATP-bound form, and modifies the local topology of the DNA (ahh, DNA topology is complicated: major groove, minor groove, writhe, twist, supercoiling, toroidal wrap), so that the helicase DnaB can bind. There's variation between species, both in the DnaA binding elements and in DnaA structure, but it remains clear that cooperativity assists in recruting enough protein to oriC to help unwind the DNA.
DnaA has four domains - domain 1 assists in interactions between DnaA and itself, as well as with DnaB. Domain II is a poorly conserved linker, domain III possesses ATPase activity as well as the primary oligomerization determinants, and domain IV binds the DnaA boxes.
There are primarily two ways that E. coli regulates replication firing at oriC. The first is sequestration, which the next paper is about - briefly, GATC sites normally exist in a fully methylated state, except when replication first occurs, after which these sites become hemimethylated due to semi-conservative replication. SeqA binds to these hemimethylated site and prevents DnaA binding. Interestingly, SeqA also forms a filamentous oligomeric structure.
The second is regulation of DnaA availability/activity. The datA locus, found somewhat close to oriC, binds DnaA with much more affinity, and deletion of this locus promotes overinitiation, suggesting that this region helps as a sink for excess DnaA. Many genes also contain DnaA binding regions that are involved in influencing transcription. Notably, this occurs at the dnaA gene itself - the sites are fully occupied and repressive only in the presence of ATP-DnaA.
There's also RIDA, or regulatory inactivation of DnaA. Hda (a homologue of DnaA) associates directly with the sliding clamp of DNA polymerase III and promotes DnaA ATPase activity.
Interestingly, bacteria have histone-like proteins that seve similar roles in DNA compaction/organization into the nucleoid, and these factors (HU, Fis, IHF, H-NS) are required for initiation synchrony by introducing bends into DNA.
SeqA: A Negative Modulator of Replication Initiation in E. coli
Lu, et al. Cell 1994
One of the ways that bacteria can regulate replication (and make sure it doesn't recur to give too many copies) is through methylation of GATC sites by Dam methylase.
This group first created/identified mutants in seqA, and developed an assay to determine how long GATC sites remained hemimethylated. They found that seqA mutants methylate GATC sites upstream of oriC quicker than wild-type, but other sites remained the same.
Then, they developed an assay to count the number of replications initiated by blocking cell division. They found that seqA mutants had all kinds of odd numbers of initiations. Also, the mutants grew abnormally, which the authors deduced was a result of excess replication forks getting stalled and inducing the SOS response.
The MatP/matS Site-Specific System Organizes the Terminus Region of the E. coli Chromosome into a Macrodomain
Mercier, et al. 2008 Cell
Background: Just like in eukaryotes, bacterial DNA needs to be highly condensed in order to fit inside the cell and efficiently segregate during cell division.
Some previous studies seem to suggest that the E. coli chromosome is ordered into 4 macrodomains (Ori, Left, Right, and Ter) with interspersed nonstructured regions. This group identified a signature sequence matS that appears numerous times throughout Ter that may assist with packaging, via a protein binding partner named MatP.
To identify a sequence that appeared often enough to serve as a strong candidate, they used a word-search type program, where they searched the genome for 11-word long repeats and used statistics to narrow down on a few that were common in Ter but rare in other macrodomains. They used this to come up with a 13-mer consensus sequence, and found that this same sequence also appeared in other bacteria.
Then, they used electrophoretic mobility shift assays (EMSAs) to observe that something binds to matS. So, they set up a screen to identify a gene responsible for a protein to bind to matS (basically, if the gene is present, it will bind to matS in a promoter region for a construct and confer streptomycin resistance), and they called it matP. Once they purified and characterized MatP, they showed its binding to matS using EMSA and calculated the affinity of the interaction and compared to MatP affinity for other sites.
For in vivo studies, they used ChIP with anti-MatP to identify sites. They extended this by doing ChIP-on-chip. Deletion of MatP lead to severe defects in chromosome segregation. Using flow-cytometry, they saw that matP deletion mutants had a few more replication initiations (rifampicin runout experiments - rifampicin binds RNA polymerase) and took longer to replicate than wild-type.
Here's a cool assay: They used attL-attR sites inserted at various parts of the genome to determine the recombination frequency between different macrodomains. They saw that matP mutants had a higher rate of recombination between Right and Ter. Next, they used a GFP-MatP fusion to visualize MatP localization in vivo. Goes from the new pole, to midcell, and before division, splits in two, and then the two foci move away. They also inserted parS/parM-mCherry sites in various macrodomains, and attempted to visualize near/co-localization with MatP.
Ok, again, I have to stop for now. These Cell papers are just WAY too long X_X.
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