References
1) Brock Biology of Microorganisms fifteenth edition Madigan, M. T., Martinko, J. M., Dunlap, P.V., Clark, D.P., Pearson Benjamin Cummings, 2018.
2) Microbiology an introduction, Tortora, G.J., Funke, B.R., Case, C.L., Pearson Benjamin Cummings, 2007.
3) The Prokaryotes, third edition, Volume1-7, Dworkin, M.
(Editor in chief), Springer, 2006
BIY 210 GENERAL MICROBIOLOGY II BIY 210 GENERAL MICROBIOLOGY II
MICROBIAL MOLECULAR BIOLOGY
Key macromolecules that carry and transmit information in prokaryotic and eukaryotic cells are,
DNA
RNA
Proteins
The flow of genetic information at the
molecular level takes place in three stages (central dogma).
replication
Transcription
translational
STRUCTURE AND FUNCTION OF THE DNA
Deoxyribose
1. C purine (adenine guanine) and primidine (cytosine, thymine)
Base + sugar = nucleoside
Deoxyribose 3.C 5.C phosphodiester
Sugar + base + phosphate nucleotide
G≡C, A=T by hydrogen bonds
Supercoil
Negative supercoils in
prokaryotes and eukaryotes Hyperthermophilic positive supercoils in archaea
Negative supercoil by twisting the right arm of the DNA double strand in the opposite direction to the axis,
In the opposite direction, a positive
supercoil is formed.
Negative supercoil is common in the non- proliferative prokaryotic genome (except hyperthermophilic archaea) and eukaryotic chromosomes.
In genome replication, negative and positive supercoils occur in prokaryotes and eukaryotes.
Catenate (intertwined in replication) and knots are also formed in the annular genomes.
Why is the supercoiling important?
In bacteria, chromosomal DNA is much
larger than the cell and fits into the cell in this way.
Supercoiling count (how many turns) is controlled by enzymes.
The level of supercoiling is not fixed. It may vary in response to environmental stress
and cellular processes (transcription, replication, and recombination).
Reactions in many genes and cells can be affected by this change. There may be
major phenotypic changes.
Topoisomerase
These enzymes, which are responsible for the formation of the supercoil and the opening of the coil, are divided into two groups.
Type I: Topoisomerase I (IA, IB and IC or topoisomerase I, III and V) EC: 5.99.1.2;
Archaea revers gyrase is in this group, ATP is not required
Opens the superhelix, a single cut reduces the imagination of the superhelix.
Eukaryotic topoisomerase I effective in positive and negative supercoils.
Prokaryotic topoisomerase I is only effective in negative supercoils.
Type II: (Topoisomerase II (DNA gyrase), IV and VI) EC: 5.99.1.3;
Breaks two yarns ATP is required
Once the genome is replicated, it forms a negative supercoil.
Opens negative and positive supercell as replication continues.
A single cut reduces the two fights of the supercoil.
Type-II topoisomerases
DNA gyrase and topoisomerase (Topo) IV, which are effective in the negative and positive supercoils, are commonly found in bacteria.
Topoisomerase II (DNA gyrase);
It creates a negative supercoil in DNA packaging in the cell.
Opens the positive supercell in replication.
Bacterial topoisomerase IV;
Opens the negative and positive supercoils in replication,
Catenane and knots s both creates and makes ring.
In most bacteria, both enzymes coexist. Some
(Corynebacteria, Campylobacter jejuni, Deinococcus
radiodurans, Treponema pallidum and some Mycobacteria) do not have Topo IV. Only gyrase is present and the activity of topo IV occurs in the cell.
In the hyperthermophile archaea, the reverse gyrase enzyme produces a positive supercoil in genome packing.
It is ineffective in topo IV genome replication in
bacteria with linear genomes (Borrelia burgdorferi and Streptomyces spp.). Streptomyces also has the gene encoding this enzyme, replication of the ring plasmids catenane formation in the early (also has linear plasmids).
DNA without topoisomerases cannot normally be replicated.
Topoisomerase inhibitors are used as anti-cancer drugs to stop the proliferation of tumor cells.
The size of a DNA molecule is
expressed as the number of bases or base pairs in the molecule.
1000 base kilobases (1 kb)
Kilobase pair (1 kbp) if DNA is double stranded
A DNA strand with 5000 base pairs is 5 kbp in size.
The E. coli genome is 4640 kbp
(4.64 megabase pairs = Mbp).
DNA polymerase
There are five types of DNA polymerases in E. coli, DNA polymerase I, II, III, IV and V.
DNA polymerase I removes RNA
primers from the DNA chain with 5 ' 3' exonuclease activity. DNA
polymerase III cannot do this.
The polymerase activity of DNA Polymerase III is higher than the
others. Therefore, it uses this enzyme
in cell replication.
DNA polymerase III
It is a complex holoenzyme.
The core part of the enzyme consists of alpha (α),
epsilon (ε) and theta (θ) subunits. This enzyme, which shows activity in double-stranded DNA, shows the
highest activity in the form of dimer (the enzyme in both strands comes together) in the replisome.
In this structure of the enzyme;
2 cores,
2 (β) beta subunits (ring subunit through which double stranded DNA passes, β sliding clamp),
2 (τ) tau subunits and
There are (γ) gamma, (δ) delta, (δ ’) delta prime, (ψ) psi and (χ) chi subunits called clamp-loading complex.
Correction of DNA replication errors
DNA polymerase III demonstrates exonuclease activity as well as
conducting DNA synthesis. With this activity, DNA polymerase III plays a proofreading role in DNA replication.
The exonuclease activity of DNA polymerase III adds to the correct
structure by removing the wrong base in DNA synthesis.
This feature also has the DNA polymerase I enzyme.
3' 5‘
Replication of Linear DNA
The primer is irreplaceable in eukaryotes, bacteria, plasmids and virus replication with linear genomes.
All DNA polymerases can add nucleotides to the 3’- OH terminal. Linear DNA does not contain a free 3’- OH lead at the site of the primer.
This problem has been solved in some viruses by circularizing the genome or forming concatemers.
Different ways have been developed to solve this problem in organisms with linear genomes.
Replication of Linear DNA
The linear DNA ends in eukaryotic chromosomes contain guanine-rich sequences called telomere, usually formed by repeating 6 base pairs for 20 or more. Replication is
achieved by binding the telomerase enzyme, which carries a short RNA as a cofactor, to the 3 ’end of the DNA to be
replicated.
Bacteria, plasmids and viruses primarily use a protein
instead of RNA. DNA polymerases can add nucleotides to the OH groups in these proteins. Proteins are covalently bound to the 5 ’ends of DNA.
Bacteria with linear genomes: Some species of Streptomyces and Borrelia genera.
Streptomyces chromosomal DNA is linear about 8 Mbp in length. Genome ends have terminal reverse repeats (TIRs) covalently linked to proteins.
Two-way replication starts from the region of origin in the middle of the chromosome and two chromosomes of similar size are formed. DNA has a terminal protein at the 5 ′ end.
Viruses with linear genomes: Bacillus subtilis phage φ29 is a model phage for linear DNA replication in prokaryotic cells. Adenoviruses in eukaryotic cells are models. DNA has a terminal protein at the 5 ′ end.
Plasmids with linear genomes: Two genera with linear genomes and some bacteria with circular genomes. Streptomyces linear plasmid is in the form of a racket with terminal repeats. The hairpin structure is seen in the Borrelia plasmid.
