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Lecture 14
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DNA replication
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DNA replication is a semi-conservative process.
One strand serves as the template for the second strand.
DNA replication is initiated at a region on a chromosome called an
origin of replication.
An enzyme called DNA Helicase binds to the origin and unwinds
the DNA in both directions from the origin.
As the DNA is unwound, specific single stranded DNA binding
proteins prevent the strands from reannealing.
RNA primers are produced by primase, which bind to the DNA.
The DNA polymerase then binds and begins to synthesize the DNA
complementary to the parental strand
DNA replication has two requirements that must be met:
1. A DNA template.
2. A free 3' -OH group.
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DNA Replication Types
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(i) Conservative mode
This hypothesis states that during replication both the strands of the
DNA remains with the parent cell and the newly synthesized two
strands of DNA is retained by the daughter cell. However this
hypothesis is not accepted anymore.
(ii) Dispersive mode
Replication would involve fragmentation of the parent DNA strands
and intermixing with the newly synthesized strands. However this
model is discarded since it not proved experimentally.
(iii) Semi conservative mode
Here the replication process of a parent DNA double helix results in
two hybrid DNAs. Here each of the daughter DNA double helices
retains a parent strand and a newly synthesized strand. The
experiments of Mathew Messelson and Franklin W. Stall has proved
the semi conservative mode of replication in the DNA.
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Semiconservative replication
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In a cell, DNA replication begins at specific locations in the genome,
called "origins". Unwinding of DNA at the origin, and synthesis of
new strands, forms a replication fork.
In addition to DNA polymerase, the enzyme that synthesizes the
new DNA by adding nucleotides matched to the template strand, a
number of other proteins are associated with the fork and assist in
the initiation and continuation of DNA synthesis.
Because DNA polymerase can only synthesize a new DNA strand in
a 5' to 3' manner, the process of replication goes differently for the
two strands comprising the DNA double helix
Nucleic acids can only be synthesized in vivo in a 5'- to 3'-direction,
as the polymerase used to assemble new strands must attach a new
nucleotide to the 3'-hydroxyl (-OH) group via a phosphodiester bond
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The replication fork
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The leading strand is that strand of the DNA double helix that is
oriented in a 5' to 3' manner. On the leading strand, a polymerase
"reads" the DNA and adds nucleotides to it continuously. This
polymerase is DNA polymerase III (DNA Pol III) in prokaryotes
The lagging strand is that strand of the DNA double helix that is
orientated in a 3' to 5' manner. Because of its orientation, opposite
to the working orientation of DNA polymerase III which is in a 3' to 5'
manner, replication of the lagging strand is more complicated than
that of the leading strand
On the lagging strand, primase "reads" the DNA and adds RNA to it
in short, separated segments, forming Okazaki fragments
DNA polymerase I "reads" the fragments, removes the RNA and
replaces the RNA nucleotides with DNA nucleotides. DNA ligase
joins the fragments together.
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Ribonucleic acid (RNA)
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Crick's central dogma.Ribonucleic acid (RNA) was discovered after
DNA.
DNA, with exceptions in chloroplasts and mitochondria, is restricted
to the nucleus (in eukaryotes, the nucleoid region in prokaryotes).
RNA occurs in the nucleus as well as in the cytoplasm (also
remember that it occurs as part of the ribosomes that line the rough
endoplasmic reticulum).
Ribosomes are 2/3 RNA (a type of RNA known as ribosomal RNA or
rRNA) and 1/3 protein.RNA was involved in protein synthesis.
Information flow is from DNA to RNA via the process of transcription,
and thence to protein via translation.
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The central dogma
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Ribonucleic acid (RNA)
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Transcription is the making of an RNA molecule off a
DNA template.
Translation is the construction of an amino acid
sequence (polypeptide) from an RNA molecule.
RNA has ribose sugar instead of deoxyribose sugar.
The base uracil (U) replaces thymine (T) in RNA.
Most RNA is single stranded, although tRNA will form a
"cloverleaf" structure due to complementary base
pairing.
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RNA types
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messenger RNA (mRNA) "Blueprint" for protein synthesis that is
transcribed from one strand of the DNA (gene) and which is
translated at the ribosome into a polypeptide sequence.
ribosomal RNA One of the three types of RNA; rRNA is a
structural component in ribosomes
Ribosomal RNA (rRNA) is the construction site where the protein
is made
transfer RNAs (tRNAs) Small, single-stranded RNA molecules
that bind to amino acids and deliver them to the proper codon on
messenger RNA.
The trucks of protein synthesis that carry the specified amino acid
to the ribosome. Abbreviated tRNA.
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Transcription
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transcription The synthesis of RNA from a DNA template.
The making of RNA from one strand of the DNA molecule
RNA polymerase opens the part of the DNA to be transcribed.
Only one strand of DNA (the template strand) is transcribed.
RNA nucleotides are available in the region of the chromatin (this
process only occurs during Interphase) and are linked together
similar to the DNA process.
During transcription, an enzyme that attaches to the promoter region
of the DNA template, joins nucleotides to form the synthesized
strand of RNA and detaches from the template when it reaches the
terminator region.
template strand The strand of DNA that is transcribed to make
RNA
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The Genetic Code: Translation of
RNA code into protein
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The code consists of at least three bases.
To code for the 20 essential amino acids a genetic code must
consist of at least a 3-base set (triplet) of the 4 bases.
If one considers the possibilities of arranging four things 3 at a time
(4X4X4), we get 64 possible code words, or codons (a 3-base
sequence on the mRNA that codes for either a specific amino acid
or a control word).
The genetic code consists of 61 amino-acid coding codons and
three termination codons, which stop the process of translation.
The genetic code is thus redundant, for example, glycine coded for
by GGU, GGC, GGA, and GGG codons.
If a codon is mutated, say from GGU to CGU, is the same amino
acid specified?
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The Genetic Code
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Protein Synthesis
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Prokaryotic gene regulation differs from eukaryotic regulation.
Promoters are sequences of DNA that are the start signals for the
transcription of mRNA. Terminators are the stop signals. mRNA
molecules are long (500- 10,000 nucleotides).
Ribosomes are the organelle (in all cells) where proteins are
synthesized.
They consist of two-thirds rRNA and one-third protein.
Ribosomes consist of a small (in E. coli , 30S) and larger (50S)
subunits.The length of rRNA differs in each.
The 30S unit has 16S rRNA and 21 different proteins. The 50S
subunit consists of 5S and 23S rRNA and 34 different proteins.
The smaller subunit has a binding site for the mRNA. The larger
subunit has two binding sites for tRNA.
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Ribosome
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Transfer RNA (tRNA)
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Transfer RNA (tRNA) is basically cloverleaf-shaped.
tRNA carries the proper amino acid to the ribosome when the
codons call for them.
At the top of the large loop are three bases, the anticodon, which is
the complement of the codon.
There are 61 different tRNAs, each having a different binding site for
the amino acid and a different anticodon.
For the codon UUU, the complementary anticodon is AAA.
Amino acid linkage to the proper tRNA is controlled by the
aminoacyl-tRNA synthetases.
Energy for binding the amino acid to tRNA comes from ATP
conversion to adenosine monophosphate (AMP).
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Transfer RNA (tRNA)
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Translation
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Translation is the process of converting the mRNA codon
sequences into an amino acid sequence.
The initiator codon (AUG) codes for the amino acid Nformylmethionine (f-Met).
No transcription occurs without the AUG codon. f-Met is always the
first amino acid in a polypeptide chain, although frequently it is
removed after translation.
The intitator tRNA/mRNA/small ribosomal unit is called the initiation
complex.
The larger subunit attaches to the initiation complex. After the
initiation phase the message gets longer during the elongation
phase
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Translation
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New tRNAs bring their amino acids to the open binding
site on the ribosome/mRNA complex, forming a peptide
bond between the amino acids.
The complex then shifts along the mRNA to the next
triplet, opening the A site.
The new tRNA enters at the A site. When the codon in
the A site is a termination codon, a releasing factor binds
to the site, stopping translation and releasing the
ribosomal complex and mRNA.
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Termination
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Often many ribosomes will read the same message, a structure
known as a polysome forms. In this way a cell may rapidly make
many proteins.
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