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5BBG0205 Molecular Basis of Gene Expression (5BBG0205)
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Kings College London (KCL)
In depth notes 5BBG0205 Molecular Basis of Gene Expression including all content from all lectures in addition to extra detail. Notes written after rewatching the lectures, and areas where content wans't clear extra detail was done and added on. FAQ that were asked, were implemented into the conten...
5BBG0205 Molecular Basis of Gene Expression (5BBG0205)
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L02: Transcription and processing of eukaryotic genes
• There are around 2m of DNA in each cell
• Crick’s dogma
o For information to flow from DNA to protein, it needs an intermediate
called RNA
o Protein can never go to DNA
• DNA bases: adenine, guanine, thymine, cytosine
o a- t= 2 hydrogen bonds
o c-g = 3 hydrogen bonds
• RNA has an additional OH group in the sugar backbone making it more reactive
and less stable compared to DNA
• RNA bases: adenine, uracil, guanine, cytokine
o Uracil lacks a methyl group
• DNA dependent RNA polymerase sits on the DNA reads the structure and
hybridizes complementary RNA bases making RNA which can be taken to the
cytosol and make protein
• DNA has a promoter and a transcript end signal
• The sequence between promoter and end signal is the gene
• Mammals have 3 polymerases; they have 5 common subunits and 10 subunit
core
o Pol1 – copies ribosomal RNA – 50%
o Pol 2 – copies messenger RNA –3%- as well as snRNA, eRNA, lincRNA
o Pol 3- copies transfer RNA – 15%
• Hence crick dogmas only accounts for 3% of all RNA produced in mRNA and
codes for protein
• RNA has protomer and poly A signal (PAS)
• Genotypes is identical between cells but the phenotypes is different
• There are 3 phases in transcription: initiation, elongation and termination
o Initiation is associated with capping
o Elongation is associated with making it through the genomes and splicing
o Termination is associated with cleavage and polyadenylation
• Transcription initiation depends on protein binding DNA and pol II
• Protein-DNA binding is based on electrostatic and hydrostatic interaction. Major
groove offers more electrostatic interaction and HBB is with the minor groove
• RNA Polymerase II (the one responsible for transcription) has a very long
extension on C-terminal domain (CTD).
• The CTD has 52 repeats of heptad amino acid sequence (YSPTSPS) - required
for post transcription modification
• CTD aids the recruitment of other proteins by polymerase.
• Promoters are not very specific. In some promoters there’s a TATA box.
, o As the promoter is not very specific its easy for pol II to not recognize a
promoter sequence and hence start transcription at the wrong location
• Promoter – a genetic element necessary for expression of a structural gene or
operon that is distinct from elements that regulate the expression level of the
gene
• To open the DNA there’s an inner play of general transcription factors (GTFs),
activators and DNA torsions
• Transcription initiation needs to be signaled to processing enzymes
• PIC (pre-initiation complex) = RNA polymerase II and a series of transcription
factors (TF)
• TF2D is multi subunit complex TATA-box-binding protein (TBP) and 13 TBP-
associated factors (TAFs)
• TF2D consists of two rigids loops: loop C and loop D that act like rulers
o These loops bind the initiation element and help loop A to recruit TBP
o TBP is a potent DNA binder hence when transcription is not necessary
TBP binds to Taf1 a protein that mimics DNA
• Preinitiation complex (PIC) assembly Commented [MN1]: faq asked about leve
o 1. TF2D establishes sequence specificity and controls TBP binding of detailed needed to know
o 2.TBP kinks DNA- aids the opening up of the DNA – point of no return
▪ The kinking allows for the two arms of DNA to come closer together
and to slightly under wind the DNA
• From PIC to open promoter
o TF2B is recruited to TBP
o TF2B recruits polymerase-TFIIE complex and helps open DNA more
o Another factors are recruited to prevent rebinding of promoter DNA
o In the presence of the different factors more and more bending is induced
• Transcription inititation – Need to remember
o Requires DNA kinking by TBP- highly regulated DNA binding
o Strand dislodging by TFII B
o Torsional strain by ATPase XPB
• Mechanism of transcription
o Trigger loops moves over and lead to the cleavage of the pyrophosphate
tier allowing the synthesis of the next nucleotide on the strand
o Bridge helix moves back and trigger loop comes for the next nucleotide
• First processing step in inititation: Formation of the RNA capping
1. RNA comes out of the active site and ser5 is phosphorylated
2. Addition of guanosine phosphate by guanylyltransferase
3. Addition of methyl group at position 7 making resistance for exonucleases
4. Removal of phosphate
• The RNA cap is resistant to degradation by exonucleases making stable to be
transported to the cytoplasm and be translated
2
, • During elongation there’s a change of the phosphorylation pattern from Ser5 to
Ser2 allowing different factors to associate with the CTD
o These factors are required to help the polymerase get through
nucleosomes, as nucleosomes needs to be half assembled and then be
put back together
• Elongation factors also allow for the correct and faster movement of polymerase,
ensure that there’s no protein stopping DNA polymerase
• In elongation, splicing occurs where introns are removed. Alternative splicing can
also occur and that is when certain exons are also removed
• Termination involves the addition of the polyA tail helping RNA stabilization and
removal of polymerase through an exonuclease degrading the RNA that is
behind the polymerase.
• Although the RNA has been spliced, there are still sequences before and after
the exon that do not code – untranslated regions
o 3’ UTR (Untranslated regions) has multiple elements that can be used for
regulation translational control of RNA
• In the 3’ there’s the poly A signal – AAUAAA, some poly A signals also gave an
UGUA called a distal poly A important for alternative polyadenylation
• CF1A and CF1B factors help with specificity by recognizing the elements around
the poly A signal
o The cleavage is achieved by an endonuclease that is inhibited for most of
the time that cuts the RNA. Endonuclease Xm2 degrades the RNA from
the 5’ to the 3’end and destabilizes the interaction with DNA and the
polymerase falls off
• Poly A tail is important for the stability and efficiency of the translation.
o Initially there’s the formation of a few As in a distributive reaction.
o Stimulation of CPSF and PABN makes it processive
o Contact to CPSG lost after 250As
▪ Reaction becomes distributives (inefficiency)
▪ Poly A tail in mammals- 70 A’s
▪ Poly A tail in Yeast- 250 A’s
• The age of the RNA can be measured by the number of A’s in the poly A tail, as
it can be determined if something has been degraded or not
• Poly A polymerase is not an efficient polymerase
• 3’end processing – summary
o Model assembly of the large cleavage polyadenylation complex is in many
ways analogous to the formation of the transcription initiation complex
o The yeast poly a signal is not very conserved the human contains one
minimal a AAUAAA hexameter and facultative additional elements.
cooperativity creates specificity
o Targeted activation of enzymatic activities only in the right context
3
, • After cleavage and polyadenylated the transcript, removal of the polymerase is
necessary. The removal is done through an exonuclease
o However there’s an issue since xm2 is slower than polII, 2kb per min
compared to 2.7kb per min respectively
▪ It has been found in order for xm2 to catch up a positive elongation
factor (Spt5) is removed slowing down pol II and allowing xm2 to
reach
• Xm2 degrades the RNA from the 3’ to 5’ end
• Some proteins are needed at high levels and others at low levels, hence
transcription levels need to regulated accordingly
• Genes expression is carefully controlled
o At all times in a viable cells
o In response to the function and needs of the cells
o Key regulatory points included
▪ During differentiation and development
▪ As a response to environment (food, hormones, toxins)
• Problems with gene expression can lead to disease
o Cancer- absence of transcription off switch leads to the wrong genes
being expressed and cell division
▪ Often aggravated by the fact that there’s the 3’ processing factor
expressed which will cut off 3’ untranslated regions and take away
the possibility of regulate the stability of the RNA
o Developmental diseases- mistakes early protein synthesis can lead to
organ developmental problems
o Chronic diseases- obesity. Abnormal gene expression as a result of the
wrong signals in the cell can cause inflammation and increased risk for
other diseases example cardiovascular disease
• One way by which transcription can be regulated is through promoter activation
• Many promoters have an associated upstream activating sequence (UAS) or
enhancer
• Transcription mediated through GFTs is inefficient
• Structures that form a DNA loop between an upstream activator and the proteins
at the TATA box and sometimes stabilized by proteins that form a rigid front for
example TCF
• Transcriptions is not confined to genes, some errors can occur with start signals
not being well defined resulting in long coding RNAs
o The RNAs are non-coding and stay within the nucleus
L03: Transcription factors - 2nd done
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