Nucleic Acids Flashcards
(39 cards)
Background
• 1953 – James Watson and Francis Crick introduced elegant double-helical model for DNA
• DNA can direct its own replication from monomers
• DNA contains hereditary information that controls your biochemical, anatomical, physiological and
behavioural traits
Evidence that DNA can transform bacteria
• 1928 – Frederick Griffith tired to develop vaccine
against pneumonia -> streptococcus
• He used two strains:
o Virulent (capsule) smooth-strain ->
pathogenic
o Non-virulent (a-virulent), rough strain ->
harmless
• Griffith wanted to know whether injections of
heat-killed virulent pneumococci could be used
to immunise against pneumonia
• He was surprised to find that when heat killed
pathogenic bacteria was mixed to living,
harmless bacteria, some of the living bacteria
was transformed to pathogenic bacteria
• This meant some chemical substance (DNA)
caused this heritable change
• This also meant that proteins couldn’t be the
factor that converted them, as proteins would
have been denatured when heat was applied
• This phenomenon is known as transformation – a
change in the genotype and phenotype due to
the assimilation of external DNA by a cell.
Only discovered in 1943 to be the DNA by Avery and his co-workers
• They created two cultures
o One where DNA was broken down leaving proteins
o And one where protein was broken done leaving DNA
o When the heat-killed pathogenic bacteria were mixed with harmless bacteria, the
conversion to pathogenic bacteria only occurred when the tube containing DNA and
broken down protein was used
o See next page for experiment example of this method
Further evidence that DNA is the genetic material
• Erwin Chargaff analysed the base composition of
DNA from a number of organisms
• In 1950 he reported that the base composition of
DNA varies from species to species
• He also noticed that the regularity of the ratios of
nucleotide bases
o In DNA -> the no. of adenine equalled the
thymines and the guanines equalled the
cytosines
• He developed the Chargaff rules:
1. Base composition varies between species
2. Within a species A and T bases are equal, and
G and C bases are equal
How is the genetic info contained in the DNA?
To fulfil its biological role, the following four must be
met by the genetic material
1. It must carry the genetic information from
parent cell to daughter cell and from
generation to generation
2. It must contain information for producing a
copy of itself
3. It must be chemically stable
4. However, it must be capable of mutation
Structure of DNA
• DNA are polynucleotides (polymers) = monomers of
nucleotides
• Nucleotide -> a nitrogenous base + phosphate group +
pentose (five-carbon) sugar
• Nucleoside -> above without phosphate group
• Adenine and guanine are large two fused carbon rings
-> purines
• Uracil, thymine and cytosine are smaller with a smaller
single carbon ring -> pyrimidines
• One purine always attaches to one pyrimidine ->
creates uniform length diameter
• Adenine bonds to thymine with two hydrogen bonds
• Guanine bonds to cytosine with three hydrogen bonds
• Deoxyribose (sugar) has one OH on its 5 carbon ring
• Ribose has two OH’s on its 5 carbon ring
• Sugar-phosphate forms the backbone of a helix strand of DNA
• Adjacent nucleotides are connected by phosphate group.
• The phosphate of one nucleotide is attached to the sugar of the next nucleotide
• 5 prime (5’) end of DNA molecule is where phosphate molecule is sticking out
• 3 prime (3’) end of DNA molecule is where sugar is sticking out
• The subunits of the two strands of the molecule run in opposite directions. This is
known as anti-parallel nature of DNA
DNA double helix
• Molecular architecture of DNA enables replication of genes
• Although the complementary base-pairing rules dictate the combinations of
nitrogenous bases that form the rungs of the double helix, they do not restrict the
sequence of nucleotides along each DNA strand
• 10 base pairs present in one full turn of the helix (3.4nm)
DNA Replication: The Basic Principle: Base Pairing to a Template Strand
• First hypothesized by Francis Crick and James Watson
• When a double helix replicates, each of the two daughter
molecules will have one old strand, from the parental
molecule, and one newly made strand -> semiconservative
model (see end of section for exp. evidence)
DNA Replication: A Detailed look
- E coli has a single chromosome (5 million base pairs) à 60 minutes for the cell to replicate
- Human cell (6 billion base pairs) à few hours to replicate
- Enormous amount of replication with very few errors occurs (1 per 10 billion nucleotides)
- High accuracy and speed requires over a dozen enzymes and other proteins
RNA Replication: Initiation
• The replication of a chromosome begins at particular sites called origins of replication à these are short
stretches of DNA having a specific sequence of nucleotides
• Bacteria have circular chromosomes with a single origin
• Proteins that initiate DNA replication recognize this sequence and attach to the DNA, separating the two
strands and opening up a replication ‘bubble’
• Replication of DNA then proceeds in both directions until the entire molecule is copied
• A eukaryotic cell may have hundreds or thousands of origins à multiple bubbles form and eventually fuse,
speeding up the process of replication in very long strands
• At the end of each replication bubble is a replication fork, is a Y-shaped region where parental strands of
DNA are being unwound
Enzymes and Proteins in DNA replication
• Helicase is an enzyme that untwists the double helix at the replication forks, separating the parental strands
and making them available as template strands
• After the parental strands are separated, single-strand binding proteins bind to the unpaired DNA strands,
stabilizing and keeping them from re-pairing
• The unwinding/untwisting of the DNA strand causes tension and strain ahead of the replication fork à
topoisomerase helps relieve the strain by breaking, swivelling and re-joining the DNA strands
• The unwound sections of parental DNA strands are now available to serve as templates for the synthesis of
new complementary DNA strands
• However, the enzymes that synthesize DNA cannot initiate the synthesis of a polynucleotide à they can only
add DNA nucleotides to the end of an already existing strand that is base paired with a template strand
• The initial nucleotide chain that is produced during DNA synthesis is actually a short stretch of RNA
o This RNA strand is called a primer and is synthesized by primase
o Primase starts a complementary RNA chain from a single RNA nucleotide, using the DNA strand as a
template
o The completed primer (5-10 nucleotides long) is base paired to the template strand and the new
DNA strand will start from the 3’ end of the primer
Synthesizing a New DNA Strand: Elongation
• Enzymes called DNA polymerases catalyse the synthesis of new DNA by adding nucleotides to a pre-
existing chain
• Two of these enzymes play a major role in replication -> DNA polymerase I and III
• Most of the DNA polymerases require a primer and template strand -> along which complementary
nucleotides are lined up
• In bacteria DNA pol. III adds a DNA nucleotide to the RNA primer and then continues to add nucleotides to
the growing end of the new DNA strand (500 per second in bacteria, 50 in humans)
Antiparallel Elongation
• As noted the two ends of a DNA strand are different, giving each strand directionality, like a one-way street
• In addition, the two strands of DNA in a double helix are antiparallel, meaning they are orientated in opposite
directions of each other
• Therefore, the two new strands formed during DNA replication must also be antiparallel to their template
strands
• Because of the structures of DNA polymerases, they can only add nucleotides to the 3’ prime end of a
primer or growing DNA strand, never to the 5’ end
• Thus a new DNA strand can elongate only in the 5’->3’ direction
Leading strand
• Along one template strand, DNA pol III can
synthesize a complementary strand
continuously by elongating the new DNA in the
mandatory 5’à3’ direction
• DNA poll III remains in the replication fork on that
template strand and continuously adds
nucleotides to the new complementary strand
as the fork progresses
• Strand made by this mechanism is known as
the leading strand
• Only one primer is required for DNA poll III to
synthesize an entire leading strand
Lagging strand
• To elongate the other new strand of DNA in the
mandatory 5’à3’ direction, DNA poll III must
work along the other template strand in the
direction away from the replication fork
• The DNA strand elongating in this direction
called the lagging strand
• Even though synthesis of both strands occur
simultaneously and at the same rate, it is known
as the lagging strand because synthesis is
delayed slightly relative to the synthesis of the
leading strand à enough template strand
needs to be exposed first before elongation can
begin
• Unlike leading strand (continuous), the lagging
strand is synthesized discontinuously, as a
series of segments
• These segments on the lagging strand are
called Okozaki fragments
• Whereas one primer is needed for the
leading strand, each Okozaki fragment
on the lagging strand must be primed
separately
• After DNA pol III forms an Okozaki
fragment, DNA pol I replaces the RNA
nucleotides of the adjacent primer with
DNA nucleotides
• But DNA pol I cannot join the final
nucleotide of this replacement DNA
segment to the first DNA nucleotide of
the adjacent Okozaki fragment
• Another enzyme, DNA ligase, is able to
complete this task and joins all Okozaki
fragments into a continuous DNA strand
-> acting like a glue
RNA & Protein Synthesis: The Flow of Genetic Information
• The information content of genes is in the form of specific sequences of nucleotides along strands of DNA,
the genetic material
• The DNA of an organism leads to specific traits by dictating the synthesis of proteins -> proteins are the link
between genotype and phenotype
• Gene expression is the process by which DNA directs the synthesis of proteins (via translation and
transcription)
• This is also known as The Central Dogma of Biology, which describes and provides an explanation to the
basic framework of how genetic information flows from a DNA sequence to a RNA sequence to a
synthesized protein product inside cells. The central dogma also suggests that DNA contains the information
needed to make all of our proteins, and that RNA is a messenger that carries this information to the
ribosomes, where they are synthesized. DNA -> RNA -> protein
RNA & Protein Synthesis: Basic principles of Transcription and Translation
• Genes provide the instructions for making a protein but a gene does not build a protein directly
• The bridge between DNA and protein synthesis is RNA
• RNA is similar to DNA except that:
o It contains ribose instead of deoxyribose as its sugar
o It contains the nitrogenous base uracil rather than thymine
o It is a single strand
• Transcription is the synthesis of RNA using information in the DNA
o The information is simply transcribed from one DNA to RNA in the form of triplet codons
o DNA serves as the template strand to assemble the complementary RNA strand
o The RNA is thus a transcript of the instructions that code for a specific polypeptide (mRNA)
• Translation is the synthesis of a polypeptide using information in the mRNA
o There is a change in language -> the cell must translate the nucleotide sequence of an mRNA
molecule into the amino acid sequence of a polypeptide -> codons match with anticodons
o The sites of translation are ribosomes, molecular complexes that facilitate the orderly linking of amino
acids
RNA & Protein Synthesis: Transcription and Translation in bacteria and eukaryotes
• Major difference in translation and transcription between eukaryotes
and bacteria is the fact that in bacteria there is no nuclear envelope
that separates DNA from protein-synthesizing equipment
• Therefore, in bacteria, the lack of compartmentalization allows for
translation and transcription to occur simultaneously. In eukaryotes,
however, the nuclear envelope prevents this from happening
• The transcription of a protein-coding eukaryotic gene results in pre-
mRNA and further processing yields functional mRNA, that travels
into the cytoplasm -> the initial RNA transcript is more generally
termed a primary transcript
Protein Synthesis: A Detailed View:
Molecular components of Transcription
• mRNA is transcribed from the template strand of a
gene
• An enzyme called RNA polymerase pries the two
strands of DNA apart and joins together RNA
nucleotides complementary to the DNA template
strand, thus elongating the RNA polynucleotide
• Like DNA polymerases that function in DNA
replication, RNA polymerases can assemble a
polynucleotide only in its 5’->3’ direction
• Unlike DNA polymerases, RNA polymerases are able
to start a chain from scratch, they don’t need a primer
• The specific sequences of nucleotides along the DNA
strand mark where transcription ends and begins
• These are known as the promotor and terminator
sequences
• The stretch in between these two sequences is
known as the transcription unit
• Bacteria have a single type of RNA polymerase whilst
eukaryotes have at least three (the one used to make
pre-mRNA is RNA pol II)
• There are three major stages of transcription are:
initiation, elongation and termination
Protein Synthesis: Initiation
• The sequence of DNA where RNA polymerase II attaches and
initiates transcription is known as the promotor
• Promotor (small string of DNA) contains within itself the start point -> place where transcription actually starts eg. TATA
Box
• In eukaryotes transcription factors (proteins) mediate the
binding of RNA polymerase to the promotor and the initiation
of transcription
• In bacteria the RNA polymerase binds directly to the promotor
• Only after transcription factors are attached to promoter does
RNA polymerase II bind to it
• The whole complex of transcription factors and RNA
polymerase II bound to the promoter is called a transcription
initiation complex
• Once the appropriate transcription factors are firmly attached
to the promotor DNA and the polymerase is bound in in the
correct orientation, does the enzyme unwind the two DNA
strands and begins transcribing the template strand at the
start point
Protein Synthesis: Elongation
• As RNA polymerase moves along DNA, it untwists 10-20
DNA nucleotides at a time for pairing with RNA nucleotides,
thereby, elongating the RNA in the 5’->3’ direction
• The enzyme adds nucleotides to the 3’ end of a growing RNA
molecule as it continues along the double helix
• In the wake of the advancing wave of RNA synthesis, the new
RNA molecule peels away from its DNA template, and the
DNA double helix reforms
• A gene can be transcribed simultaneously by several
molecules of RNA polymerase -> this helps cell make the
protein in large amounts
Protein Synthesis: Termination
• In bacteria transcription proceeds through a terminator sequence in the DNA. The transcribed
terminator (an RNA sequence) functions as the termination signal causing, polymerase to detach from
DNA and release the transcript, which requires no modification before translation
• In eukaryotes, RNA polymerase II transcribes a sequence on the DNA called the polyadenylation signal
sequence, which specifies a polyadenylation signal (AAUAAA) in the pre-mRNA. This is called a signal
because once this stretch of 6 RNA nucleotides appears, it is immediately bound by certain proteins in
the nucleus. The proteins then cut it free from the polymerase, releasing the pre-mRNA. The pre-mRNA
then undergoes processing. However, the RNA polymerase continues to transcribe. Since the new 5’
end isn’t protected by a cap, enzymes degrade it. The polymerase continues to transcribe until the
enzymes catch up to dissociate it from the DNA.
Modification of pre-mRNA: in Eukaryotic cells
• Before being sent to cytoplasm the transcript is modified and undergoes RNA processing by enzymes in the
nucleus
• During this processing, both ends of the RNA transcript are altered
• Also some interior sections of the RNA molecule are cut out and the remaining parts spliced together.
Alteration of mRNA ends
• The 5’ end is synthesized first and receives a 5’
cap (modified form of guanine)
• The 3’ end is modified before mRNA exits the
nucleus, an enzyme adds 50-250 more
adenine nucleotides, forming a poly-A-tail
• Both of these serve important functions:
1. Facilitate the export of the mature mRNA
from the nucleus
2. They help protect the mRNA from
degradation by hydrolytic enzymes
3. They help ribosomes attach to the 5’ end of
the mRNA once it reaches the cytoplasm
• The ends aren’t translated and neither are UTR
(untranslated regions) -> they assists in
ribosome binding
