Structure and roles of DNA, RNA
--- complimentary base pairing
 no conservative replication (no hybrids
1 nucleotide =
Pentose + nitrogenous base + phosphoric acid
RNA
- single stranded (except genomes of some viruses)
- mRNA, tRNA, rRNA = involved in protein synthesis
2nd generation cells:
Half the cells had hybrid DNA, while the other half
had pure 14 N-DNA
DNA is packed in chromosomes. Each DNA associates
with proteins called histones. DNA is slightly
negative in electronegativity. Histones are positively
charged. Thus DNA and histones associate via ionic
attractions.
 Excludes dispersive replication (no pure
obtained)
Most DNA is wound around outside of groups of 8
histones to form nucleosomes. Remaining is linker
DNA, joining adjacent chromosomes. Nucleosomes
and linkers constitute the chromatin fibre.
- Before start of DNA replication: free nucleotides
are manufactured in cytoplasm, transported into
nucleoplasm via nuclear pores
1)
2)
3)
5 carbon sugar, RNA – ribose (OH),
DNA – deoxyribose (H)
phosphoric acid (h3PO4), when dissociated, it
gives nucleic acids its acidic character.
nitrogenous bases- purines/ pyramidines
a. purines – double ring- adenine, guanine
b. pyramidines – single ring – cytosine,
thymine (uracil in RNA)
Nucleotide Formation = Condensation rxns
nitrogenous base attaches to 1C of sugar
phosphoric acid attaches to 5C of sugar
two water molecules are removed
Nucleic Acid Formation = Condensation rxn
nucleic acids formed by combining
nucleotides (condensation btw phosphate
group of one with sugar of other)
covalent bond linking adjacent nucleotides
called phosphodiester bond
long polynucleotide chain has backbone of
alternating sugar and phosphate groups
with bases projected sideways frm sugar
sugar-phosphate backbone
“Anti-parallel”
5’: phosphate group attached to sugar 5C
3’: hydroxyl group to sugar 3C
polynucleotide has 5’ to 3’ orientation
Chargaff’s Rules
1) ratio of A:T = 1: ration of G:C =1
2) ratio of (A+G): (C+T) = 1
 width btw 2 sugar phosphate backbones = o.2nm =
combined width of a pyramidine + purine
 1 complete turn of the double helix has 10 base
pairs, 3.4nm
2 strands held in position by weak hydrogen bonds
forming btw nitrogenous bases of opposite strands
 Adenine pairs thymine => 2 H bonds
 cytosine pairs guanine => 3 H bonds
Start of DNA Replication
1.
Further coiling  chromosome
2.
Semi-conservative replication: Both strands of DNA
separate and each act as a template for the
synthesis of a new complementary strand.
Each DNA formed Is a hybrid consisting of one old
and one new strand (semi-conserved)
Conservative replication: parental double-helix acts
as template for synthesis of a new DNA double helix,
Parental DNA remains intact and goes into one
daughter cell while newly synthesized DNA goes into
other daughter cell.
Dispersive: Parental strand fragmented and
dispersed to daughter strands. Each strand of both
daughter molecules contains mixture of old and
newly synthesized parts.
3.
4.
5.
6.
7.
Results of Meselson-Stahl experiment
1st generation cells:
DNA bands in caesium chloride were located
between pure 15N -DNA and pure 14 N -DNA. Thus,
DNA of first generation cells was a hybrid (1 14 N
strand, 1 15N strand)
N DNA
Semi- Conservative Replication
- occurs during S-phase of eukaryotic cell cycle
Chromatin fibre coils around itself to form solenoid,
6 nucleosomes per turn of the helix in a solenoid.
Describe Process of DNA replication, evidence for
semi-conservative replication
14
8.
replication begins at origins of replication
(specific sequence of nucleotides)
specific enzymes (helicases, topoisomerase)
and other proteins initiate replication.
They bind to each origin of replication on
parental DNA molecule.
Helicase/topoosomerase disrupts H bonds
between complementary base pairs.
Ensures 2 parental strands of double helix
unwind and separate, replication forks
form and spread in both directions
creating replication bubble.
Once a primer is in place, the enzyme
DNA polymerase catalyses the elongation
and synthesis of the complementary
daughter strand.
The end of the RNA primer provides free 3’
OH which is required for DNA polymerase
to initiate DNA synthesis.
DNA polymerase recognizes the bases,
selects free deoxyribonucleotides to be
aligned in a sequence complementary to
parental strand.
DNA polymerase catalyses formation of
phosphodiester bonds between adjacent
deoxyribonucleotides of the newly formed
daughter strand.
As DNA polymerase moves along template,
part of enzyme proof-reads (check if
proper base pairing is made, swiftly
remove incorrect deoxyribonucleotide,
replace with correct one)
9.
DNA polymerase links deoxyribonucleotides
to growing daughter strand frm 5’ to 3’.
10. a different DNA polymerase removes RNA
primer, replacing it with DNA
-
1.
2.
Since parental strands are anti-parallel, 2
daughter strands synthesized in opposite
directions.
Leading strand is synthesized continuously
in 5’ to 3’ direction,
Lagging strand also same direction, but in
shorter segments of 100 to 300 nucleotides.
Fragments produced by this discontinuous
synthesis are called Okazaki fragments.
Each okazaki fragment initiated by RNA
primer before addin deoxyribonucleotides
Okazaki fragments extend in 5’ to 3’
direction and eventually join up with other
fragments forming continuous DNA strand.
continuous DNA strand when DNA
polymerase excises RNA primer and
replace it with DNA
a linking enzyme ligase joins 3’ of each
new DNA fragment to 5’ of growing chain
by forming phosphodiester bond.
Transcription carried out by RNA polymerase in
Nucleolus
b.
Translation performed on ribosomes in cytoplasm
c.
DNA code  mRNA
Sequence of bases on mRNA molecule
complementary to that on DNA template it was
derived from.
Triplet bases in mRNA called codons.
Code is degenerate, non overlapping, not
punctuated, bordered by start [(AUG) and stop
(UAG/UAA/UGA) codons, universal.
Starts/stops polypeptide chain synthesis during
translation
d.
e.
SO…
RNA Polymerase moves from 3’ to 5’
mRNA synthesized in 5’ to 3’ direction.
f.
Protein Synthesis
Ribosomes interact with mRNA and amino-acidcarrying transfer RNA molecules, to translate info in
mRNA molecule into polypeptide.
region, forming transcription factor-DNA
complex.
Complex recognized by RNA polymerase,
binds to promoter region.
RNA polymerase unwinds DNA doble helix
at initiation site. DNA has 2 exposed
strands, sense strand is transcribed
(template) for mRNA production
Free ribonucleotides from nucleoplasm
matched with DNA template by
complementary base pairing (A-U)
RNA polymerase catalyses joining of
adjacent ribonucleotides through
formation for phosphodiester bonds.
g.
When RNA polymerase reaches end of gene,
polymerase dissociates and newly formed
RNA released. Common termination
sequence AATAAA.
Pre-mRNA processed before leaving via
nuclear pores. Splicing = excision of
introns (non-coding regions), join exons.
mRNA (3-5%) of total RNA in cell, single-stranded
RN formed from DNA template during transcription
Let’s talk about RIBOSOMES.
The sequence of nucleotides in the DNA molecule
determines order in which amino acids are joined
together to form proteins.
rRNA (80%), combines with protein to form large
and small ribosomal subunits, existing via nuclear
pores. In cytoplasm, subunits combine to form
functional ribosomes. (synthesized in nucleolus)
Gene = specific sequence of nucleotides along DNA
molecule, coding for specific sequence of amino
acids in a polypeptide chain
tRNA (15%)folded back upon itself, held in shape by
H bonding between
Central Dogma
= flow of genetic info from DNA to protein
1.
2.
3.
4.
genetic info stored as DNA in all cells, in
many viruses too. Replication of genetic
info involves DNA synthesis
genetic info expressed in cell, flowing
directionally from DNA to RNA
RNA to protein (translation)
reverse transcription possible (RNA to DNA)
Replication is carried out by DNA polymerase in
nucleolus
There is anticodon region  made of specific
triplet base sequence located at specific region on
folded tRNA molecule – complimentary to codon
foud on mRNA
3’ end of tRNA always ends with CCA, where amino
acid specific to tRNA binds.
-
aggregates of protein and rRNA.
Eukaryotes [40s small subunit, 60S large
subunit = 80S ribosome]
P site = peptidyl tRNA site
A site = aminoacyl-tRNA site
E site = exit site
TRANSLATION is… mRNA  amino acid sequence
Amino Acid activation
Amino acid attached to specific tRNA. Reaction
erquires ATP, catalysed by group of enzymes called
aminoacyl-tRNA synthetases
tRNA + amino acid = aminoacyl-tRNA complex.
1. TRANSCRIPTION
DNA sequence of a gene is used as
template to make complementary base
sequence of mRNA.
a. Transcription factors bind to promoter
Binding of mRNA to ribosome
Small subunit attaches to 5’ end of mRNA. Slightly
downstream from 5’ end is AUG.
Initiation of polypeptide chain
Initiator aminoacyl-tRNA binds to AUG start codon
MRNA, aminoacyl-tRNA, small ribosomal subunit is
joined by large ribosomal subunit.
Initiator aminoacyl tRNA positioned in P site of large
ribosomal unit. GTP provides energy for assembly of
this complex.
2nd level of specificity – matching of specific
anticodon to codon.
Chain elongation
Codon Recognition
anticodon of 2nd aminoacyl-tRNA complex
forms H bonds with second codon on mRNA
through complementary base pairing. This
is held at A site of ribosome.
Hydrolysis of 2 molecules of GTP provides
energy for this step.
Peptide bond formation
peptide bond formed between 1st and 2nd
amino acid (catalysed by peptidyl
transferase, present in large ribosomal
subunit
first a.a. dissociates from tRNA.
Translocation
ribosome shifts one codon down in 5’ to 3’
direction. 2nd aminoacyl-tRNA complex
now from A site to P site. 2nd now exit site
1st tRNA is now released to cytoplasm for
recycling.
A site ready to receive 3rd aminoacyl-tRNA
comlex with anticodon complementary to
third codon along mRNA.
So. P site holds the tRNA carrying growing
polypeptide chain, while A site holds tRNA carrying
next amino acid.
Chain termination
Specific proteins called release factors enter A site
instead, causing hydrolysis of bond between
polypeptide chain and tRNA in P site. Polypeptide is
released from ribosome, to complete folding to
assume secondary/tertiary structure.
Ribosome disassembles into subunits.