Download coding region of DNA. o Introns – non

30 Gene expression: Transcription
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Gene structure.
o Exons – coding region of DNA.
o Introns – non-coding region of DNA.
o Introns are interspersed between exons of a single gene.
o Promoter region – helps enzymes find the correct starting point for translation. Translation
initiation occurs before the START codon in exon 1.
o Terminator region – includes regulatory sequences that are important but do not contribute
to the protein. Translation finishes after the STOP codon in the final exon.
o Un-translated regions (UTR) (blue) – transcribed sequences that are not translated.
 5’ UTR and 3’ UTR are important but not used to encode for amino acids.
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DNA strands.
o DNA template strand.
 Strand used to generate complementary mRNA sequence.
 Either DNA strand can act as a template strand.
 Direction of gene is always from 5’ to 3’ on the coding strand. But the direction of
the gene is 3’ to 5’ on the template strand.
 Both strands do not encode the same gene.
o DNA coding strand.
 Strand that is similar to mRNA except that U is replaced by T.
 Not used in transcription.
 Contains the order of the amino acid sequence.
mRNA direction.
o RNA transcription always occurs in a 5’ to 3’ direction.
o Reverse complement – mRNA is always complementary to DNA template strand.
Prokaryotes.
o All genes are transcribed by a single RNA polymerase.
o Made of 5 subunits (core - 𝛼2 𝛽𝛽′𝜔) with a detachable sigma (𝜎) factor (holoenzyme).
 Core enzyme is required for polymerisation activities.
 Holoenzyme is required for binding to the promoter region (correct initiation of
transcription).
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Prokaryotic promotor binding (step 1 of transcription).
o Sigma factor binds to the promoter region to determine where RNA synthesis should begin.
Forms the closed complex.
o RNA polymerase switches to an open complex. It “melts” the double-stranded DNA to form a
transcription bubble.
o Once the core enzyme is bound, the sigma factor dissociates.
o 𝜎 70 is the primary sigma factor, but there are other sigma factors for different purposes.
o Function of each subunit.
 Alpha – determine the DNA to be transcribed.
 Beta – catalyse polymerisation.
 Beta’ – bind and open DNA.
 Omega – unknown
 Sigma – recognise initiation sites (promoter regions).
o Consensus sequences.
 Highly conserved sequences in the promoter region that help the sigma factor find
the start of genes.

Mutations in these sequences can result in the gene not being expressed.
 -35 and -10 (bp from start of transcription) regions (separated by 15-17 bp) in the
promoter for E. coli.
 TATA box = -10 region, sequence happens to be TAT and generally followed by AAT.
Eukaryotic promotor binding (step 1 of transcription).
o Eukaryotes have 3 RNA polymerases used for transcription.
 RNA polymerase I – rRNA (5.8S, 18S and 28S).
 RNA polymerase II – mRNA, some snRNA.
 RNA polymerase III – tRNA, rRNA (5S), some snRNA.
o Promoters contain sequences that determine the specificity of the type of RNA pol binding.
o Most promoters contain:
 Upstream regulatory elements.
 TATA box.
 Transcriptional start site.
o
General transcription factors.
 Necessary for the initiation of transcription – bind promoter and facilitate RNA
polymerase II binding.
 Undergo sequential binding with polymerase.
 TFIID binds to the TATA box.
 TFIIA and TFIIB subsequently bind.
 The complex is then bound by RNA polymerase, on which TFIIF is already attached.
 A pre-initiation complex is formed by the binding of TFIIE and TFIIH.
 TFIIH has ATPase and is responsible for unwinding the DNA helix and separating the
two strands.
 Following ATP dependent phosphorylation, TFIIH forms the transcription bubble and
RNA polymerase can now initiate transcription (without transcription factors).
o
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Specific transcription factors.
 Two types: activator and repressor proteins.
 Activator proteins bind to enhancer regions further upstream either proximal (close
by) or distal (many bp away).
 Whether the STF are present or not determines if a given cell will initiate
transcription or not.
 They cause the DNA to fold as the specific transcription factor binds to the initiation
complex via mediators and co-activators.
 This interaction increases the rate of transcription.
 When a repressor protein binds to a silencer sequence which is adjacent to or
overlapping an enhancer sequence, the activator protein cannot bind to the DNA.
o These lead to high regulation of eukaryotic transcription, and thus is key to differential gene
expression.
Genetic elements that regulate transcription.
o Tissue-specific transcription factors.
o Repressors present in some regions and absent in others.
Elongation (step 2 of transcription).
o RNA polymerase breaks interactions with transcription factors and escapes the promoter
region to start elongation.
o RNA polymerase moves along the DNA template strand and adds bases in the 5’ to 3’
direction of the growing RNA strand.
o Bases are complementary to the DNA template.
o RNA polymerase binds to ~30 DNA bp at a given time.
 ~14 bp are unbound by RNA (in a transcription bubble).
 ~12 bp are bound as a RNA-DNA hybrid region.
o There is progressive proof-reading, so it is possible to back up and correct mismatches.
o Multiple RNAs can be transcribed simultaneously.
Termination (step 3 of transcription).
o Prokaryote termination - simple.
 RNA polymerase encounters chain termination sequence with high G-C content
followed by at least 4 Us.
 Resulting RNA transcript is self-complementary and causes a hairpin to form with a
stem and loop structure.
 RNA and RNA polymerase dissociates from the DNA.
o Prokaryote termination – rho dependent.
 DNA template contains a signalling sequence that is made of inverted repeats and is
40 bp long.
 The mRNA sequence has a transcript of this sequence that is called the rho
utilisation site (rut).
 Rho is an ATP-dependent helicase that binds to the rho utilisation site and moves
along the RNA (requires energy).
 The terminator sequence in the DNA template causes the RNA polymerase to slow
down.
 When the rho protein catches up to the RNA polymerase, it initiates termination of
the RNA polymerase.
o
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Eukaryotic termination.
 Different for each RNA polymerase.
 RNA polymerase II.
 Passes the sequence 5’AAUAAA3’.
 The cleavage and polyadenylation specificity factor (CPSF) binds to the
sequence.
 A number of other factors including a cleavage stimulating factor and
cleavage factor proteins also bind to form a complex.
 This complex causes the mRNA to cleave.
RNA processing in eukaryotes.
o RNA is still in the nucleus.
o 5’ capping.
 Addition of a 7-methyl guanosine cap.
 Caps protect the growing RNA from degradation by nucleases.
 Recognised by translation machinery.
o 3’ polyadenylation.
 Facilitated by poly(A) polymerase.
 Addition of up to 200 adenine bases in the form of a Poly(A) tail.
 Enhances mRNA stability and regulates transport to cytoplasm.
o RNA splicing.
 Removal of introns.
 Primary transcript is spliced.
 Exons are joined up to make the final transcript.
 Small nuclear RNAs (snRNA) joined with proteins form small nuclear
ribonucleoproteins (snRNP).
 snRNPs recognise the boundaries between introns and exons, in which they can
recognise a number of different sequences.
 snRNPs associate to the start and end of an intron that needs to be spliced out.
 They then interact with each other to form a spliceosome, causing the intron to
form a loop.
 The spliceosome then cuts at the splice site and ligates the exons together.
 The intron that has been cut out is degraded in the nucleus.
o
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Alternative RNA splicing.
 Can result in different proteins from the same primary RNA transcript, which may be
more suited to the cellular needs in different tissues/organs.
 Types:
 Exon skipping – cutting out an exon by including it in the loop.
 Mutually exclusive exons – integrate either one of two middle exons.
 Alternative 5’ donor sites – Can begin splicing in the middle of an exon,
removing the latter part of the exon.
 Alternative 3’ acceptor sites – Can end splicing in the middle of an exon,
removing the previous part of the exon.
Drosophila sex determination.
o Homodimers that result from the two X chromosomes in the female are transcription factors
for the gene that produces the Sxl (sex-lethal) protein.
o It binds to the promotor in the Sxl gene in females, and so the Sxl protein is made only in
females.
o The Sxl protein is important in coordinating the splicing in the next gene in the sequence,
which is the Transformer protein gene (Tra).
o There is a functional Tra protein in females, but not males.
o The Tra protein promotes the use of an alternate splice site to skip a particular exon that
codes for the male DSX (DSX-M) protein.
o The result is a DSX-F protein in females for female differentiation and a DSX-M protein in
males for male differentiation.