LOs Wk 12
Wine Dark Sea
Gene Expression and Regulation
Describe how the process of gene expression leads to the synthesis of a specific protein.
Gene expression is how genes make their products at the right time and in the right place. Gene expression is
tissue-specific; only certain genes are active and being expressed for a specific tissue type. Gene expression
occurs in two phases, transcription and translation.
Transcription (in cell nucleus) is the synthesis of RNA under the direction of DNA. Both nucleic acids use the
same language, and the information is simply transcribed, or copied. Just as DNA strand provides a template for
the synthesis of a new complementary strand during DNA replication, it provides a template for assembling a
sequence of RNA nucleotides.
RNA polymerase attaches to the promoter region of the DNA and the DNA strand begins to unwind. The
polymerase initiates RNA synthesis at the start point on the template strand. The polymerase moves downstream
in a 5’ -> 3’ direction elongating the RNA transcript. At the terminator the polymerase detaches from the DNA
and the RNA transcript is released. This is the pre-mRNA strand. This newly formed strand contains both exons
(coding regions) and introns (non-coding). The non-coding regions are spliced forming the mRNA transcript. This
mRNA is a faithful transcript of the gene’s protein-building instructions.
Translation (in cell cytoplasm) is the actual synthesis of a polypeptide, occurs under the direction of mRNA in
the ribosomes. During this stage there is a change in language. The cell must translate the base sequence of an
mRNA molecule into the amino acids sequence of a polypeptide.
Active mRNA enters the cytoplasm and is transported to the ribosomes (RER, or free ribosomes). The ribosome
binds to the 5’ end of the mRNA and the process of tRNA binding and amino acid attachment begins. tRNA
molecules have a specific anticodon at one end and a corresponding amino acids at the other. This process
continues along the mRNA codon triplets until the amino acid chain is complete and the protein backbone is
constructed.
Demonstrate, using the example of thalassemia, how mutations in DNA sequence can alter the
expression, sequence and function of proteins.
A gene mutation is a permanent change in the DNA sequence that makes up a gene. Mutations can be germ-line
(hereditary) or somatic (acquired). Mutations vary greatly and can result in codon deletion, frame shifts, and
premature stop of translation or translation of inappropriate proteins, resulting in a change or loss of normal
function.
-Thalassemia: are associated with deletions that result in reduced or absent synthesis of -globin chains.
There are 4 genes present producing -globin, the more genes deleted the greater severity.
β-Thalassemia: a mutation of the β-globin gene, most commonly at the promoter region (transcription
terminated), during slicing (inappropriate active mRNA) or of the chain terminator (creates a new stop codon
within an exon, either an insertion or a deletion, resulting in a frameshift mutation). Sequencing of β-thalassemia
genes has reveled more than 100 different causative mutations, mostly consisting of point mutations.
The two causative mutations of β-Thalassemia fall into two categories:
1. βo mutations: absent β -globin synthesis. Most commonly associated with a chain terminator mutation.
2. β+ mutations: reduced, but detectable β-globin synthesis. Most commonly associated with splicing
mutations.
As a result of the impaired gene expression due to a mutation, the β-globin synthesis is reduced or absent.
Thalassemia mutations
SPLICING MUTATIONS: (most common cause of β+ mutations) most of these mutations lie within introns, while a
few are located within exons. Some of these mutations destroy the normal RNA splice junctions and completely
prevent the production of normal β-globin mRNA, resulting in βo -thalassemia. The functional protein is
LOs Wk 12
Wine Dark Sea
destroyed. Others create an ‘ectopic’ splice site within an intron. Because the flanking normal splice site remains,
both normal and abnormal splicing occurs and some normal β-globin mRNA is made, resulting in β+-thalassemia.
PROMOTER REGION MUTATIONS: these mutations reduce transcription by 75-80%. Some normal β-globin is
synthesised, thus these mutations are associated with β+-thalassemia.
CHAIN TERMINATOR MUTATIONS: these are the most common cause of βo –thalassemia. Two subtypes of
mutations fall into this category. The most common type creates a new stop codon (set of three bases) within an
exon; the second introduces small insertions or deletions that shift the mRNA reading frames. The altered reading
frame reads a different genetic message, causing an entirely new series of amino acids to be coded after the site
of mutation.
Both block translation and prevent synthesis of any functional β+–globin.
Describe the regulation of gene expression with particular reference to transcriptional and
translational control.
Transcriptional Control- transcription factors
There are a number of control elements that regulate the initiation of transcription by binding transcription
factors. These may be located proximal or distal to the promoter. Distal control elements are enhancers.
Transcription factors have a DNA binding domain and an activation domain. The activation domain bind other
proteins of the transcription machinery that result in gene transcription. Transcription factors include inducers
(activate) of specific genes and repressors (inactivate) of specific genes.
For example, RNA polymerase creates a complementary strand of the DNA and on reaching the terminator, the
polymerase detaches and releases the RNA. This RNA is know as pre-mRNA and must undergo further processing
to become functional mRNA. Pre-mRNA contains both exons and introns. The introns (the non-coding regions) are
sliced leaving the exons, which combine to form the active functional mRNA.
Translational Control- mRNA stability; protein stability.
Targets for translational regulation include initiation factors, mRNAs, and ribosomes. Translational control
functions to stabilize mRNA and allows the creation of a final stable functioning protein (splicing).
RNA processing: the final protein is controlled via alternative RNA splicing, where different mRNA molecules are
produced from the same primary transcript. Regulation proteins determine whether they need to splice an exon
or an intron.
mRNA degradation: for specific protein expression, mRNA may have a limited life span in response to
environmental changes and is thus degraded. mRNA breakdown is stimulated by a enzymatic shortening of the
poly-A tail. This triggers enzymes to remove the 5’ cap, allowing nuclease enzymes to destroy the mRNA.