Download File - Wk 1-2

Gene Expression and Regulation
1. Describe how the process of gene expression leads to the synthesis of a specific protein
 Gene expression is regulated so that a cell produces only those proteins that are
needed, thus, at any time only a subset of the available genes will be expressed.

Gene expression depends on the type of gene:
- Constitutive Gene expression/positive regulation – encodes for genes that are
constantly required and always turned on.
- Inducible gene expression/negative reglulation – encodes for genes that are
made in response to the environment and are able to be turned on/off.
Inducible gene expression is modulated by inducer proteins which turn it on
and repressor proteins which turn it off.
Positive regulation – is when the bound modulator turns on the gene and leads
to mRNA transcription.
Negative regulation – is when the bound modulator turns the gene off and
prevents mRNA transcription

The primary function of gene control is to adjust the enzymatic machinery of the cell
to its immediate nutritional and physical environment
Concentration of a protein depends on:
 Synthesis of the primary transcript – transcription from DNA and splicing
 Post-translational processing of mRNA – poly A tail and capping
 mRNA degredation – binding to the 5’ or 3’ ends to help stable the mRNA
 Protein synthesis (translation) – translation on the ribosome and successful folding
 Post-translational modification of proteins – phosphorylation, glycosylation for
perfect folding or function
 Protein degradation – proteins have a half-life as a way of regulating/turning them
off/killing them
 Protein targeting and transport – specific targets e.g. ER, mitochondria, and external
membrane. Must be in the correct spot in order to function properly. This also
regulates gene expression
2. Demonstrate, using the example of thalassaemia, how mutations in DNA sequence can
alter the expression, sequence and function of proteins
Proteins are derived from DNA. If the gene nucleic acid is altered in any way then the
protein will be altered
Mutation – is a permanent, transmissible (germ-line or somatic) change in the nucleotide
sequence of a genome, which leads to a change/loss of normal function.
Mutations can vary in size from 1bp - > 100kbp and via codon deletion, alteration or phase
shift and they can cause premature stop of translation, translation of different proteins,
translation of ineffective proteins etc.
α-Thalassaemia results from the recombination between sister chromosomes, which causes
either the deletion of one or more α-globin genes, or an increase in the number of genes
(from 2 to 3)
β-Thalassaemia generally results from mutation of the β-globin gene, rather than gene
deletion. Mutation may occur at the areas such as the promoter (no initiation of
transcription), the cap-site (no protection from hydrolytic enzymes), in the process of
splicing (altered active mRNA) etc. These will result in impairment of gene expression and
reduced β-globin production.
3. Describe the regulation of gene expression with particular reference to transcriptional
and translational control. Indicate the function of the promoter, introns and exons

Gene expression occurs in two basic steps:
o Transcription – synthesis and processing of RNA
o Translation – synthesis and processing of protein
TRANSCRIPTION
 Within the nucleus of the cell, RNA polymerase II which is highly specific for certain
promoter regions attaches to DNA at the promoter and travels along the strand to
the terminator, producing a strand of RNA complementary to the template as it
goes.
 On reaching the terminator, the polymerase is released and the RNA dissociates.
This RNA is now known as pre-mRNA and must undergo further processing before it
is functional
 A 5’ 7-methylguanine cap and a 3’poly A tail are added – these protect the
RNA from enzymatic degradation, promote movement into the cytoplasm
and act as an attachment point for ribosomes.
 Pre-mRNA contains both exons (coding regions) and introns (non-coding
regions) – the latter are removed by splicosomes leaving the exons, which
combine to form the active mRNA transcript
TRANSLATION
 Active mRNA enters the cytoplasm and is transported to ribosomes on the RER or
free ribosomes. The ribosome binds to the 5’ end of the mRNA and the process of
tRNA binding and amino acid (AA) attachment begins. This process continues along
the mRNA condon triplet until the AA chain is complete and the protein backbone is
constructed. Several ribosomes can act on the same mRNA sequence producing
several proteins at once.
 Post-translational modification occurs once the protein is complete and may involve
phosphorylation, glycosylation etc which may be vital to the function of the protein.
The protein can then be transported to its site of action to carry out its functions.
 A gene is not fully expressed until its protein product is transported from its site of
synthesis to its site of action (protein targeting) and if necessary, converted to a
functionally active protein (protein processing).

On reaching its correct destination, it may need to associate with other proteins in
order to form a functional unit e.g. multi-subunit enzyme complex (protein
assembly)
Promoter – the sum of DNA sequences necessary for transcription initiation. They’re
located just upstream from the site where transcription begins.
Enhancers – are DNA sequences that regulate transcription from a distance.
Both promoter and enhancer sequences form binding sites for regulatory proteins that
either stimulate or repress transcription.
4. Outline briefly the steps in transcription, RNA processing and translation
TRANSCRIPTION – transfer of information from DNA’s base sequence to the complementary
base sequence of the mRNA. Essentially the same information is relayed
1. Transcription factor stimulates histones at the transcription site and binds to the
promoter. Promoter – the sum of DNA sequences necessary for transcription
initiation. They’re located just upstream from the site where a transcription/mRNA
synthesis begins. The promoter also decides which DNA strand becomes the
template.
2. Transcription factor also acts on RNA polymerase which is the enzyme that manages
synthesis of mRNA.
3. RNA polymerase binds to the template and unwinds 16-18 pairs of DNA bases at a
time.
4. It uses ribonucleoside triphosphates as a substrate and aligns them with the
complementary DNA base pairs (e.g. AGC – UCG)
5. When the polymerase codes a termination signal/stop codon (UAA, UAG, UGA)
transcription is terminated
TRANSLATION – In the cytoplasm, information carried in the mRNA is decoded and
constructed into polypeptides.
1. mRNA molecule binds to rRNA. tRNA then transfers AA’s dissolved in cytosol to the
ribosome.
2. Approx 45 tRNA exist that bind to specific AA’s. This attachment is controlled by
synthetise and is activated by ATP.
3. tRNA helps to manoeuvre the AA into the correct position as specified by mRNA
4. One end of tRNA (the stem) is bound to the AA. The other end (the head) is bound
to the anti-codon
5. The anti-codon is a 3-base sequence that is complementary to the mRNA strand. E.g.
mRNA codon is UUU (Phe), the tRNA’s carrying Phe will have the anti-codon AAA.
6. For translation to commence, the mRNA attaches to a small ribosomal subunit by a
‘leader sequence’ of bases. It’s only function is to make this attachment. At the
same time, an initiator tRNA also attaches to a ribosomal subunit. These 2 subunits
attach together and form a functional ribosome.
7. The ribosome then scans along the leader sequence until it reaches a start codon
(AUG) which is recognised and bound by the anti-codon (UAC) of the tRNA. This
process requires energy from guanosine triphosphate (GTC)
8. Initiator tRNA binds to the P site (middle portion) of the ribosome and the 2 sites
either side of the P site are vacant (E and A). Once the codon is matched with its
anti-codon, the tRNA moves along to site E.
9. As the mRNA chain continues to move through the ribosome, tRNA with its anticodon head and AA stem, attach to the codons and produce a growing protein chain.
10. The initial portions of mRNA which have already been read may move through other
ribosomes so multiple copies of the same protein can be produced. This mRNA
complex is called polyribosome.
11. This translation continues until a STOP codon (UDA, UAA or UAG) is reached.
5. Describe common types of mutations
Somatic Mutations
- Happen in a non-germ line cell
- Organism becomes a mosaic
- Most common causes of cancer
Germ-line Mutations
- Occurs in cells that give rise to gametes
- Gametes will carry the mutation into the next generation
- Germ-line mutations are heritable
POINT MUTATIONS (Substitutions)
A base pair substitution - the replacement of one nucleotide and its partner from the
complementary strand with another different pair of nucleotides
Two types:
 Transition: Substitution of one pyramidine by the other, or one purine by the other
(e.g. T→C or A→G); more frequent. (NB: pyramidines = CUT, purines = AG)
 Transversion: Purine ←→ pyramidine
Point mutations are caused by:
 Malfunction during the replication of DNA causing the wrong base to be inserted
into a polynucleotide chain during DNA synthesis
 Chemical modification of DNA – directly changes one base pair into a different base
Some point mutations have no effect as the substitute base pair still codes for the same AA
due to the redundancy of the genetic code. Other substitutions may code for different AA’s
and still have little or no effect while some single substitutions may have great effect as in
the case of cystic fibrosis.
The substitution may be in the binding site and alter the proteins ability to bind substrate or
impair proper protein folding. Point mutations are usually missence meaning the altered
codon still codes for an AA whether it be the right or wrong one.
Nonsense mutations are those which change the AA codon to a stop signal, leading to nonfunctional proteins
FRAME SHIFT MUTATIONS: (insertions and deletions)
Alters the subsequent reading frame by inserting or deleting one or more bases (within a set
of three). This alters the reading frame (triplet grouping) of the genetic message, causing an
entirely new series of AA’s to be coded after the site of the mutation.
All nucleotides downstream of the mutation will be improperly grouped into codons
resulting in extensive missense mutations and sooner or later a nonsense mutation. These
mutations usually have more disastrous effects than substitutions on the resultant protein.
Source of inserted materials lies with transposable elements (sequences of DNA with the
ability to move from one site to another)
Frame shift mutations are caused by:
 Mistakes made during replication or recombination
 Treatment of DNA with intercalating agents (e.g. acridine orange, proflavine)
Deletions of part or all the inserted material, and sometimes the adjacent regions, may
subsequently revert by loss of the inserted material; a deletion cannot be reverted
SUPPRESSOR MUTATIONS:
When a second mutation overcomes the deleterious effects of a first mutation you can see
reversion to original phenotype
 Second mutation within same codon leading back to mutation of original AA
 Second mutation within same codon, but specifying different AA with new AA,
restoring the function of the protein
 Second mutation within same gene but different codon, with second mutation
counteracting first and restoring original function
 Second mutation within a different gene (intergenic suppressor mutation)
SPLICE SITE MUTATIONS
Mutations that erase or change the site of intron and exon splicing. Causes large scale
alterations in a protein sequence and basically destroys functionality of proteins. They can
be caused by point or frame shift mutations.
The mutations mentioned above only involve one or a few nucleotide base pairs. However
mutations do occur over larger sequences. Mutations may be large (>100kbp), medium (10100kbp) or small scale (1-100kbo). Larger mutations are obviously deleterious to functional
protein production.
Mutational consequences
Classifications of mutations:
1. Gain of function
- Where the product does something which is absolutely abnormal
- Needs to be a much more specific type of mutation
2. Loss of function (most common)
- the product has reduced or no function
- for most genes, the precise amount is not critical – such as in most inborn errors in
metabolism
-
however in cases where 50% of the normal level is insufficient for normal function,
haploinsufficiency produces and abnormal phenotype (humans are normally diploid)
occasionally the mutant allele interferes with the function of the normal allele giving
a dominant negative effect
Many different changes in a gene can cause the loss of function of the gene or its protein
1) Delete
o Part of the gene (Duchenne’s muscular dystrophy)
o The entire gene (α thalassaemia)
2) Insert a sequence into a gene
o LINE-1 repetitive sequence into FVIII gene in haemophilia A
3) Disrupt the gene structure
o By translocation
o By inversion
4) Prevent the promoter working
o By mutation
5) Destabilise the mRNA
6) Prevent correct mRNA splicing
o Alteration to normal splice site
o Alteration to splicing enhancers or suppressors
o Activation of cryptic splice site
7) Introduce a frameshift in translation
o Changes function rather than destroys function
8) Convert a codon to a stop codon
9) Replace an essential AA
o E.g. haemoglobin and sickle cell anaemia
10) Prevent post-transcriptional processing
11) Prevent correct cellular localisation of product
12) Epigenic modification without DNA sequence change (imprinting)
6. Indicate how point mutations may alter function: regulation, sequence, premature
termination, post-translational modification, trafficking
See Q 5 above.
7. Explain why most mutations result in recessive alleles