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Lec1: microbial genetics
Ass.Prof. Dr. maysa salih
Genetics: Is the study of the inheritance or heredity of living
things
A genome is a complete set of genetic material (genes) in a cell.
For example, an ovum or a sperm contains a genome. The
genetic material of living things is predominantly DNA.
However, the genetic material of some viruses such as HIV and
Ebola is RNA.
A gene can be defined as a sequence of DNA which codes for
the synthesis of one polypeptide.
Bacterial chromosome
Consists of a single molecule of double-stranded
deoxyribonucleic acid (DNA) in ring shape which is in
association with histone like proteins. A bacterium contains one
chromosome. It contains hereditary information which is passed
from one generation to the next generation. The procaryotic
chromosome is not surrounded by a nuclear membrane. A
bacterium may contain one or more extra piece of chromosomes
called plasmids. Plasmids are circular,double-stranded DNA.
Plasmids may contain genes responsible for antibiotic
resistance. They have been used as vectors to transfer the
foreign genes into the bacterial cells in genetic engineering
techniques.
DNA consists of building blocks called nucleotides. A
nucleotide consists of a phosphate, a sugar (deoxyribose) and a
nitrogenous base (adenine, guanine, cytosine and thymine).
Nucleotides link together to form a polynucleotide (strand).. A
DNA consists of two strands arranged in an anti-paralleled
direction. If one strand goes from the 3' to 5' direction, the other
strand always goes in the 5' to 3' direction. These two strands are
held together by hydrogen bonds between their nitrogenous
bases according to the base-pairing rules: adenine with
thymine, and cytosine with guanine. The number of hydrogen
bonds between adenine and thymine is 2, and that between
cytosine and guanine is 3.
DNA REPLICATION:
DNA replication is the process in which DNA is duplicated. In
E. coli, the process takes about 20 minutes
The Overall Replication Process:
The process involves the following basic steps: (1) uncoiling of
DNA, (2) unzipping of the hydrogen bonds so each strand
serves as a template for the synthesis of a new strand, and
(3)synthesis of a new strand whose bases are complementary to
those of the old strand.DNA synthesis is described as semiconservative type because each old strand is used as a template
for the synthesis of a new strand. So, in every DNA molecule
one strand is always an old one and another strand is always a
new one.
The first stage of DNA replication in prokaryotes is the
uncoiling of the DNA double helix by the enzyme helicase.
Helicase separates the DNA into two template strands. RNA
primase then adds a short sequence of RNA to the template
strands. This short sequence of RNA is a primer which allows
DNA polymerase III to bind to the strands and start the
replication process. Once this is done, DNA polymerase III adds
nucleotides to each template strand in a 5'→3' direction. The
nucleotides have 3 phosphate groups and are called
deoxyribonucleoside triphosphates. Two of these phosphate
groups break off during the replication process to release
energy. Since the strands are anti-parallel (the two strands have
their 5' end and 3' end in opposite sides) and replication can only
occur in a 5'→3' direction, one of the strands will be replicated
in the same direction as the replication fork and the other will be
replicated in the opposite direction of the replication fork. This
means that one of the strands is synthesised in a continuous
manner (named the leading strand) while the other one is
synthesised in fragments (named the lagging strand). The
leading strand only needs one primer while the lagging strand
needs quite a few as it is formed in fragments. These fragments
are called Okazaki fragments. DNA polymerase I will remove
the RNA primers and replace these with DNA. The enzyme
DNA ligase then joins the Okazaki fragments together to form a
continuous strand.
TRANSCRIPTION: THE FIRST STAGE OF GENE
EXPRESSION
Only one strand of DNA is transcribed into mRNA. The strand
that contains base sequence for synthesis of protein is called the
sense strand (template strand). The strand that does not
contain information for the synthesis of protein is called the
nonsense, noncoding or antisense strand
PROTEIN SYNTHESIS
Transcription Translation
DNA ------------------ > mRNA ----------------- > Protein
The segment of DNA that contains the information for the
synthesis of a protein is called a gene.
Transcription:
The process of synthesizing RNA based on the sequence of
nucleotides in DNA is called transcription. It involves three
phases: initiation, elongation and termination.
Initiation: The initiation of transcription begins with the
attachment of RNA polymerase on specific locations
(sequences) on DNA molecule called promoters. The
promoters determine which DNA strand (sense strand) will be
transcribed. The enzyme contains a subunit called the sigma
factor (a protein). The sigma factor determines which promoter
site on DNA will begin transcription. Once the core of RNA
polymerase attaches to the promoter site, the sigma factor
dissociates from the enzyme. In most cases, the triplet on DNA
that begins transcription is TAC which is transcribed as AUG
(codes for methionine) on mRNA.
Elongation:
The segment of DNA that contains genetic information for a
specific protein is uncoupled by RNA polymerase. Free
ribonucleotides (ATP, UTP, CTP and GTP) are automatically
attracted by the exposed bases on one polynucleotide strand (the
sense strand) which contains the genetic information. New
nucleotides are linked to the exposed bases by hydrogen bonds
according to the base-pairing rules: adenine pairs with uracil, AU; thymine pairs with adenine, T-A; and cytosine pairs with
guanine, C-G. The RNA polymerase catalyzes condensation
reaction between the newly attached nucleotides by the
elimination of inorganic pyrophosphate, forming a new
polyribonucleotide which is called messenger -RNA (mRNA).
The direction of RNA synthesis is similar to that of DNA,
starting from the 5' end to the 3' end of the mRNA. As the
mRNA is still being synthesized, its 5' end detaches from the
DNA and immediately attaches to the ribosome to initiate
translation. In eucaryotes, the mRNA has to move out the
nucleus through nuclear membrane before translation can begin.
When the mRNA breaks away from the DNA, the two
polynucleotide strands of DNA rejoin together.
Termination:
A specific molecule called the rho protein moves along the
sense strand. When it encounters the termination sequence, it
signals to the RNA polymerase to end the transcription process.
The mRNA then breaks away from the DNA
Translation involves three steps:
1. Initiation
2. Elongation
3. Termination
Initiation:
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5’ end of mRNA binds to the small subunit of the ribosome
initial mRNA codon = AUG = start codon
tRNA with anticodon: UAC binds to mRNA AUG codon by
complementary base pairing, methionine attached to tRNA 3’
terminal
large ribosomal subunit binds, completing ribosomal structure, and
producing two ribosomal binds sites: P site & A site
B. Elongation:
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tRNA with anticodon complementary to second mRNA codon
binds to ribosomal A site, with appropriate amino acid attached to
tRNA 3’ terminal
enzymes in ribosome catalyze formation of peptide bond between
methionine and 2nd amino acid
P site tRNA, now separated from methionine, exits ribosome
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ribosome moves one codon (3 nucleotides) toward the 3’ end of
mRNA, thus shifting previous A-site tRNA to P-site, and opening
A-site
tRNA with anticodon complementary to A-site mRNA codon binds
to ribosomal A-site, with appropriate amino acid attached to tRNA
3’ terminal
enzymes in ribosome catalyze formation of peptide bond between
2nd and 3rd amino acids
P site tRNA, now separated from its amino acid, exits ribosome
ribosome moves one codon (3 nucleotides) toward the 3’ end of
mRNA, thus shifting previous A-site tRNA to P-site, and opening
A-site
repetition of process until stop codon is reached
C. Termination:
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when ribosomal A-site reaches a stop codon, no tRNA has a
complementary anticodon
release factor protein binds to ribosomal A-site stop codon
polypeptide and mRNA are released
large and small ribosomal subunits separate