Microbiology Daily Newsletter

February 17, 2014 - Bacterial Genetics

At this point, all students should be able to provide a solid description of Replication, Transcription and Translation. Therefore, we will not concern ourselves with the basics of these processes, but instead focus on how Bacteria differ from Eukarya in these three processes. As we move though this week, we will also begin our discussion on genetic regulations.

The following topics in genetics you should have a strong familiarity with. If you don't then it is time to brush up on your genetics.

Be able to discuss replication, transcription and translation.
Be able to describe in detail the mechanisms of replication, transcription and translation.
Be able to discuss the concept of a genome, and what constitutes a genome.
Be able to discuss supercoiling of DNA and the use of topoisomerases.
Be able to discuss how DNA analysis is done, and why.
Be able to discuss how researchers analyze the whole genome, and why it is important.
Be able to discuss all of the different types of RNA.
Be able to discuss post-translational modification of proteins.
Be able to describe open reading frames, paralogs, orthologs and DNA alignments.
Be able to discuss various mechanisms of gene regulation and epigenetics.

Bacterial DNA Replication

The genetic process of replication is fundamental to all living systems, and the basic mechanism is the same: DNA is unwound and opened, each parental strand acts as a template upon which a newly synthesized strand is based; the resulting daughter molecules are semi-conservative, being formed of one template (old) strand and one new strand. You should be intimately familiar with this process.

So, what is different between bacterial replication and eukaryotic replication?
Remember that the nucleoid (genophore) of the bacteria is a single circular molecule that is vastly smaller than eukaryotic chromosomes. Unlike the chromosome, which can have multiple origins of replication, bacteria will generally only have 1 origin of replication (Ori).

Eukaryotic chromosomes are linear, and so you keep replicating til you get to the end (remember, there is a problem with the ends potentially degrading...remember telomerase?). Bacterial DNA is circular, so there is no end. Instead, you find a terminus, or termination point (Ter).

As with all DNA replication, you will get two replication forks on either side of the origin of replication, these will then meet at the terminus. All the while, the DNA is anchored to the cell membrane, and it is the movement of the cell membrane that will separate the two daughter molecules.

The process begins when DnaA binds to the Ori. DnaA, and the specific sequence at Ori, are genera/species specific. The most highly studied system is Escherichia coli, where a repeat of 5' - TTATCCACA - 3' is used as the binding sequence for DnaA.

DnaA enzymes needed for replication including Helicase, DnaB, and DnaC. DnaB anchors at what will become replication forks, and aids in the binding of primase.

Single-stranded binding proteins and DNA gyrase will be needed to prevent re-annealing and positive supercoiling respectively. DNA polymerase III holozyme will be the primary work horse of the elongation phase.

Termination is interesting, as the two replication forks are forced to meet in the Ter region, by a process referred to as the replication fork trap. The two daughter strands are joined as interlocking rings in the terminus (catenane, or mechanically linked molecular architecture). DNA Topoisomerase IV will be the critical player in unlinking the daughter molecules, and will need DNA gyrase to assist.

From: Charvin G et al. PNAS 2003;100:9820-9825. Configurations of a circular replicating DNA. (a) Model for in vivo strand separation: the progression of the replication complex leads to the formation of (+) supercoils (L-nodes) in front of the replication fork and R-precatenanes behind. Topo IV removes (+) supercoils and gyrase generates (–) supercoils so that, under the action of both enzymes, the chirality of the precatenanes is inverted. Topo IV may then unlink the molecules by removing the L-catenanes. (b) Model for in vitro decatenation by Topo IV: R-catenated plasmids may form L-supercoils that are removed by Topo IV with a high rate [as observed in our experiments and some bulk assays (21)]. However, the removal of the last few links is slow , because Topo IV will relax only a rare fluctuation of an R-node to an obtuse angle that is similar to its preferred substrate: an L-node with acute angle (see Fig. 1b).

Daily Challenge

Review DNA replication as it pertains to bacteria, and consider, how would you move from the replication of a circular DNA molecule to a linear molecule? What would need to change? Initiation? Elongation? Termination? How different are each of these process from eukaryotic replication?