8 dna replication, gene expression (v3)members.optusnet.com.au/~romainedb/notes/Lecture 8 grey.pdf · Translation and transcription are coupled in prokaryotes – no nucleus. 8 2004

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2004 Biology Olympiad Preparation Program 2

DNA REPLICATION


DNA structure

Phosphate Deoxyribose

Nitrogenous base

Nucleotide

DNA strand = DNA polynucleotide

1’

2’ 3’ 4’

5’


DNA structure

dsDNA is antiparallel.

Hydrogen bonds hold the

two chains together.

Native form of dsDNA in cells is

the double helix. It is very stable.


Semiconservative replication

Conservative – parental DNA intact, copy is entirely new.

Dispersive – daughter molecules contain a mix of old

and newly made DNA.

Semiconservative – daughter molecules contain one old, one

new strand of DNA.


Origin of replication DNA replication begins at

origins of replication.

The DNA double helix opens up to form a small bubble.

Helicase unwinds the double helix at the ends of the bubble.

Single-stranded binding proteins hold the two strands apart.


Replication bubbles and forks Replication fork – Y-shaped region

where new strands of DNA are elongating.

2 replication forks per replication bubble.

DNA replication proceeds in both directions of each replication

bubble.

Multiple bubbles speed up DNA replication. They grow and

eventually fuse.

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Prokaryote bubbles

Prokaryotes have circular chromosomes, and only have one origin of replication


Priming

Primase attaches and synthesises a short RNA

strand complimentary to one of the DNA strands.

Primase works 5’ to 3’.

Required because DNA polymerase cannot initiate its

own strand of DNA.


Elongation – leading strand

DNA polymerase adds DNA nucleotides to the 3’ end of the

RNA primer.

New DNA strand is lengthened by DNA polymerase through complimentary base pairing

with the template strand.

DNA synthesis always occurs in the 5’ to 3’

direction.

This is the leading strand.


Elongation – lagging strand

5’ 3’

5’ 3’

5’ 3’

Lagging strand cannot be made continuously – DNA polymerase can only add nucleotides to a free 3’ end.

Lagging strand is made of fragments that are linked together.

Leading & lagging strands are made at the same time.


Elongation – lagging strand Primase synthesises an

RNA primer.

DNA polymerase adds DNA nucleotides to the 3’

end of the primer.

Process continues, and Okazaki fragments are made.

Another DNA polymerase replaces the RNA primer with DNA, and DNA ligase seals gaps, forming the

completed lagging strand.


Simultaneous synthesis

Leading strand is made continuously.

Lagging strand is synthesised in fragments which are then joined.

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DNA replication


Proteins involved in DNA replication

Leading strand Lagging strand

Double helix unwinding, providing ssDNA templates

Helicase Single-stranded binding protein

Priming Primase

Elongation DNA polymerase

Replacement of RNA primer

DNA polymerase

Primase Priming for Okazaki fragment

DNA polymerase

Elongation of fragment

DNA polymerase

Replacement of RNA primer

Ligase Joining of fragments


Proof-reading DNA polymerisation DNA polymerase proof-reads each nucleotide as it is added,

against the template strand.

If it finds an incorrectly paired nucleotide, it removes it and resumes new strand synthesis.

DNA polymerase activities:

5’ 3’ polymerase – synthesis 3’ 5’ exonuclease – proof-reading

5’ 3’ exonuclease – removing primers


Repairing DNA damage Cells continuously monitor and

repair their genetic material.

Many repair mechanisms take advantage of base-pairing of

DNA.

Nucleotide excision repair (left) – endonuclease cuts, DNA

polymerase fills, ligase seals.

Mismatch repair – involves enzymes similar to NER.


The end-replication problem DNA polymerase removes the RNA primer but needs a free 3’ end from which to

polymerise the primer replacement – at the end,

this is not available.

After repeated replications, the ends

of daughter DNA strands gets increasing

shorter.


Telomeres Special repetitive

nucleotide sequences at the ends of chromosomes – prevent gene erosion.

Telomerase produces a 3’ overhang so that successive

DNA replications do not reduce overall chromosome length.

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DNA replication, in summary •  Deoxyribonucleic acid is a polymer of nucleotides. •  Nitrogenous bases in DNA are adenine, guanine, cytosine and

thymine. •  DNA is a double helix in its native form. •  Replication of DNA occurs in a semiconservative fashion –

daughter dsDNA contain one old and one new strand •  DNA replication begins at origin(s) of replication where

helicase unwinds DNA and single-stranded binding proteins hold the ssDNA strands apart.

•  Replication bubbles contain two replication forks. Eukaryotic DNA replication involves multiple bubbles, while prokaryotic involves one.


DNA replication, in summary •  Primase synthesises an RNA primer to which DNA polymerase

can add nucleotides. •  DNA polymerase adds nucleotides to a free 3’ end of the

primer by base pairing rules. •  DNA synthesis occurs in the 5’ 3’ direction. •  The leading strand is synthesised continuously. •  The lagging strand is composed of Okazaki fragments made by

primase, DNA polymerase, and sealed with ligase. It is synthesised in fragments.

•  Leading and lagging strands are synthesised simultaneously. •  DNA polymerase proof-reads what it polymerises. •  DNA polymerase has 5’3’ polymerase activity, 3’5’

exonuclease activity and 5’3’ exonuclease activity.


DNA replication, in summary •  All cells continuously monitor and repair DNA damage. •  Nucleotide excision repair is conducted by endonucleases,

DNA polymerases and ligases. •  The inability of DNA polymerase to stick nucleotides on to 5’

ends of existing nucleic acid molecules means that linear chromosomes shorten after successive replications.

•  Telomeres are a solution to this problem. They are non-encoding repetitive DNA sequences at ends of eukaryotic chromosomes.

•  Telomerase lengthens telomeres. It is found active in germ-line cells and cancerous cells.


GENE EXPRESSION


One gene – one polypeptide

Study of auxotrophs lead to the one gene –

one enzyme hypothesis.

Not all proteins are enzymes, and not all proteins are made up of

only 1 polypeptide chain. One gene – one polypeptide hypothesis.


The flow of genetic information

Transcription – DNA to mRNA

Translation – mRNA to polypeptide.

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The genetic code 20 amino acids but only 4

nucleotides!

Need 3-letter code: 43 = 64 combinations, enough to encode 20 amino acids.

Triplet code.

Template strand transcribed to mRNA, codons translated by

ribosomes to polypeptide.


The genetic code Highly conserved.

Redundant but not ambiguous.

Reading frame important:

THE CAT ATE THE RAT

HEC ATA TET HER ATX


Transcription – initiation RNA polymerase is

responsible for transcription.

Works 5’ to 3’ – ‘downstream’.

RNA pol attaches to the promoter, and transcription is ended at the terminator. DNA portion that is transcribed is the

transcription unit.

Transcription factors bind to the promoter before RNA pol

binds. 2004 Biology Olympiad Preparation Program 29

Transcription – elongation

RNA polymerase moves along the DNA, synthesising new

RNA in the 5’ 3’ direction using complementary base

pairing rules. This direction is with reference to the newly

produced DNA.

Double-helix reforms after RNA pol has passed, and RNA

strand peels away from template DNA.


Transcription – termination

Transcribed terminator sequence acts as the termination signal.

RNA pol drops off as does the newly synthesised

pre-mRNA.


Alterations of mRNA ends Coding segment codes for

polypeptide, flanked by the start and stop codons and

untranslated regions (UTRs).

Modified guanine cap attached to 5’ end, poly

adenine (poly-A) tail added to 3’ end.

Facilitate ribosome attachment, assist export from nucleus, helps protect mRNA from degradation.

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RNA splicing Removal of non-coding regions

in the coding segment.

Small nuclear ribonucleoproteins (snRNPs)

+ other proteins = spliceosome.

Introns excised, and exons ligated to form completed

mRNA.


Export

Eukaryotes make mRNA in the nucleus.

mRNA must be transported out of nucleus through

nuclear pores to cytoplasm where translation can take

place.


tRNA

Aminoacyl-tRNA synthetase

Transfer RNA (tRNA) transfers amino acids to

ribosomes.

tRNA molecules differ in their anticodon

sequence, which bind to a complementary codon on mRNA.

Amino acids are joined to their own tRNA by

aminoacyl-tRNA synthetase.


Ribosomes – in depth Ribosomes – small and large subunits,

made of protein and rRNA.

tRNA fits into binding sites when its anticodon base pairs with an

mRNA codon in that site.

P site – holds the tRNA attached to the growing polypeptide.

A site – holds the tRNA carrying the next amino acid to be added.

E site – discharged tRNAs exit here.


Translation – initiation Small ribosomal subunit binds upstream of the start codon.

Moves downstream, finds start codon (nearly always AUG).

Initiator tRNA binds to the start codon, bearing methionine.

Large ribosomal subunit binds, forming the initiation complex.

Initiator tRNA sits in the P site.


Translation – elongation Appropriate tRNA enters the A site, its anticodon base pairing

with the codon exposed in the site.

Peptide bond formation is catalysed by the ribosome.

Ribosome moves 1 codon downstream, translocating the

tRNA.

P tRNA moves to E and leaves, A tRNA moves to P, and A site is open to next tRNA.

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Translation – termination UAA, UAG, UGA act as stop codons, and do not

code for amino acids.

Release factor (a protein) binds to the stop codon in

the A site.

The translation assembly falls apart, releasing the completed polypeptide

chain.


Post-translational modification

Modifications may be needed after translation is complete to make a functional protein from the polypeptide(s).

Attachment of sugars, lipids, phosphate groups (phosphorylation), etc.

Removal of parts of the polypeptide.

Joining polypeptides together.


Polyribosomes

Once a ribosome has moved past the start codon, another one can attach – multiple ribosomes can

translate the same mRNA simultaneously.

These strings of ribosomes are called polyribosomes, or polysomes.


Signal peptides

Signal peptide targets the polypeptide for the ER. Taken by another protein to the ER. Polypeptide is fed into the ER and folds into its

final conformation.


Roles of different types of RNA mRNA Carries genetic information from

DNA to ribosomes.

tRNA Translates mRNA codons into amino acids.

rRNA Found in ribosomes.

Primary transcript Precursor to mRNA, tRNA or

rRNA, and may be processed by cleavage or splicing.

snRNA Found in spliceosomes.

SRP RNA Plays a role in signal peptide recognition.


Prokaryote vs eukaryote gene expression RNA polymerase and ribosomes

are different.

Eukaryotes rely on transcription factors to initiate transcription.

Translation and transcription are coupled in prokaryotes – no nucleus.

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Gene expression, in summary •  One gene codes for one polypeptide. •  Genetic information flows from DNA to RNA to polypeptide. •  The genetic code is redundant but not ambiguous. •  Reading frame of codons is important. •  Transcription copies an RNA message (in the form of mRNA)

from DNA. •  Transcriptional initiation requires transcription factors attaching

to the promoter before RNA polymerase binds (eukaryotes). •  Post-transcriptional modifications in eukaryotes include a 5’

modified G-cap and 3’ poly-A tail. •  Pre-mRNA is spliced in eukaryotes by spliceosomes, removing

introns and ligating exons.


Gene expression, in summary •  mRNA is exported out of the nucleus in eukaryotes before

translation can begin. •  Translation interprets the mRNA message (in codons) to

polypeptides by way of ribosomes and specific tRNA. •  Translation is initiated at the start codon, AUG, coding for the

initiator tRNA, carrying methionine. •  The ribosome catalyses peptide bond formation as tRNAs bring

amino acids to the ribosome. •  Stop codons (UAA, UAG, UGA) code for a protein release

factor that causes the translation assembly to fall apart. •  Polypeptides may undergo post-translational modifications

before becoming a functional protein.


Gene expression, in summary •  Many ribosomes can translate a single mRNA transcript at once

in polyribosomes. •  Signal peptides target polypeptides for specific destinations in

eukaryotes. •  Many types of RNA exist in cellular metabolic machinery,

especially in gene expression. •  Prokaryotic and eukaryotic RNA polymerase and ribosomes are

different. •  Prokaryotic transcription and translation are coupled.


Next time…

•  Mutations •  Mendelian genetics •  Non-Mendelian genetics

When?

Documents

8 dna replication, gene expression (v3)members.optusnet.com.au/~romainedb/notes/Lecture 8 grey.pdf · Translation and transcription are coupled in prokaryotes – no nucleus. 8 2004