Summary of Advanced Molecular Biology.
All theoretical lectures are summarized in this summary. Lecture 1 is not included, this lecture is about several techniques used for Molecular Biology research. Out of my own experience, the course was pretty hard, so start early to really grasp the informat...
• Gene structure: coding sequence, promoter, untranslated region
Coding sequence = Everything in the open reading frame that codes for RNA.
Untranslated region = The part of the DNA outside of the open reading frame, that does not get
translated. The parts outside of the promotor and terminator do not get a transcript either.
• general principles of transcription regulation (bacteria and eukaryotes)
Transcription is regulated by a few important things:
- Start Codon: ATG
- Stopping Codon: TAG, TAA, TGA
These are the two codons that are on the edges of an Open reading frame. But RNA Polymerase does
not recognise the ATG itself. It recognises a promotor (only when sigma factor is bound to it)
The Promotor:
This is a sequence which is in front of a gene. It has a specific
-35 and -10 sequence (TGACAA, TATAAT). These sequences
can be recognised by the sigma factor σ70 , which is an
important sigma factor for housekeeping genes in a
prokaryote.
This sigma factor defines the starting point, direction and
intensity of the transcription.
The terminator:
This is the after the stop codon of the gene, which tells the RNA polymerase to terminate the
process.
The transcription can be divided into three parts:
Initiation: This is the RNA Polymerase which recognizes the sigma factor on
the promotor and binds to it.
Elongation: The RNA Polymerase starts the transcription process; it reads
DNA From 3’ to 5’ and builds an RNA strand from 5’ to 3’.
Termination: The RNA Polymerase recognizes the terminator and gets
loose from the DNA. Ending the transcription process.
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,• Sigma factors,
The sigma factors are able to bind to recognize specific regions of the promotors ( -35 and -10
regions) and bind to them. RNA Polymerase can recognise this complex and initiate the transcription
process.
A few notable sigma factors:
σ70- Most common one, is used for the housekeeping genes
in prokaryotes.
σS – This factor is used when the bacterial load is too high
(too crowded) in a environment.
σ54 – this Sigma factor is regulated by an activator. This
activator DNA site is called an enhancer. σ54, is able to bind
to the activator NTrC which is able to bind to this enhancer
further away from the promotor + sigma factor.
• regulation of transcription (lac operon, one- and two-component regulatory systems)
The Lac Operon:
This is a common operon on E. Coli bacteria. It’s function is to
produce lactase, which can metabolize lactose for energy.
But producing genes takes energy, and it would be wasteful to
make genes when it is not needed. The lac operon only works when
there is lactose in the environment.
The operon consists of a promotor, CAP Site and Operator.
- If there is no lactose in the environment. The lac repressor
will bind to and block the operator. Sigma factor is not able
to bind to it.
Lactose is able to bind to the lac repressor and disconnect it from
the DNA. The operator however does not have the ideal sequences
for σ70 factor to bind. This is why the promotor of the lac operon is
called a weak promotor.
- If there is lactose and glucose in the environment. There is low cAMP in the cytoplasm. The
σ70 is able to weakly bind and create low transcription. The bacteria prefers to use glucose as
a source of energy.
- If there is no glucose on the environment, the cAMP in the cytoplasm is high. cAMP is able to
bind to CAP, which is able to bind to the CAP-site together. cAMP:CAP on the CAP-site
enhance the binding of σ70 to the promotor.
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,One- and Two component regulatory systems:
In Prokaryotes, one- and two- component regulatory systems can be distinguished. One-component
regulatory systems can have regulatory and has the sensory in functions in the same protein. Two-
component regulatory systems have separate proteins for sensory and binding/regulation.
CAP-protein (Catabolite activator protein):
This is a protein which is a one-component regulatory system. The F-helixes in the DNA-binding
domain are able to change the orientation. When they are turned in the right way, they are able to
bind to DNA.
Whenever cAMP binds to the CAP protein, the sensory c-AMP binding domain activates in the
protein and forms H-bonds with the central C-helixes. Whenever this happens the F-helixes turn in
the right way and are able to bind to DNA.
NtrC & NtrB:
These proteins together form a two-component regulatory system. The
NtrC itself can not bind to the DNA enhancer and needs to be
phosphorylated. This is done by the NtrB protein, which can sensor low
glutamine in the cytoplasm.
Whenever glutamine is not bound to NtrB, the protein will unfold;
activating the His-kinase transmitter domain. This domain is able to
activate and phosphorylate the NtrC regulatory domain. And makes it
able for the protein to bind to the DNA.
The pros of the one component system is that it is faster. The pros of the two-component system is
that it is more versatile (different combinations of proteins are possible).
This lecture only gave an introduction to eukaryotic transcription. The following learning goals are
also included in the next summary.
• RNA polymerase I, II and III, and corresponding class I, II and III genes
Eukaryotes have 3 RNA Polymerases which are able to make different
products.
RNA Pol I: Makes pre-ribosomal RNA (pre-rRNA)
RNA Pol II: Makes most of the mRNA and snRNA
RNA Pol III: Makes small RNAs.
α-amanitin is a chemical compound which is made by several fungi. It is
able to inhibit RNA Pol II.
3
, In this lecture only a few genes corresponding the RNA pol II were given. The whole RNA Pol II
complex has 12 subunits.
Bridge complex & trigger loop
These are the compounds that push the whole RNA Pol II complex forwards and the main functioning
units for the elongation process of RNA.
The trigger loop is open whenever the RNA Pol II is on a new nucleotide. Whenever it is open it is
able to insert a nucleotide onto the RNA strain. If it has completed
• CTD (RNA polymerase II), role of CTD phosphorylation
The largest subunit of the RNA Pol II protein is the C-Terminal Domain (CTD) which
is a repeat of amino-acids (YSPTSPS)n. Different parts of this domain can be
phosphorylated; which causes different readers to be attracted to the CTD and
causes the correct proteins to be recruited to the RNA Pol II complex.
The phosphorylation (by kinases) and dephosphorylation (by phosphatases)
therefore is important to transcription of RNA. One transcription cycle can have
different parts of the repeats be phosphorylated and dephosphorylated during the
elongation.
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