Detailed summary of the elective course advanced molecular biology. This summary is accompanied by a poster that provides an overview of the entire cell cycle (phases, control mechanisms, cyclins, CDKs, checkpoints etc). This information, even if you don't take this course, is really useful for oth...
Summary advanced molecular
biology
Index
Chapter 1 Transcriptional regulation I
Chapter 2 Transcriptional regulation II
Chapter 3 Transcriptional regulation III
Chapter 4 Post-transcriptional regulation: processing
Chapter 5 Post-transcriptional regulation: splicing and export
Chapter 6 Post-transcriptional regulation: cytoplasmic regulation
Chapter 7 DNA repair recombination and replication
Chapter 8 The cell cycle
Chapter 9 Cancer
Chapter 10 Experimental techniques
1
,Chapter 1 transcriptional control
of gene expression I
The amount of protein in a cell determines the cell’s functions and properties and is thus an
important target of regulation, especially to respond to environmental clues, to functionally
differentiate and homeostasis.
Regulation of proteins levels can occur at various events in the protein’s lifespan:
Transcription initiation
Elongation
RNA processing
mRNA export from nucleus
mRNA degradation
mRNA translation
protein degradation
Eukaryotes have: histone octamers, chromatin condensation, post-translational modifications of
histone tails, introns, DNA splicing
Transcriptional regulation
Transcriptional regulation is essential to let processes work without problems, and to not waste
energy by transcribing a precise, well balanced amount of protein
Transcription is divided in three phases:
- Initiation: first RNA polymerase subunit(s) assembly to promoter
- Elongation: DNA polymerase reads DNA 3’5’ and transcribes the RNA 5’3’
- Termination: stop codon
Transcription is mainly regulated at the initiation step rate limiting step thus determines the
overall rate of protein translation and consequent partially the amount of protein, regulation at
another phase has no effect (except the termination; only for transcript length however)
Initiation: start of the RNA polymerase
RNA polymerases associate with the promoter: a certain nucleotide sequence upstream of the start
of the gene (and the open-up point, denoted 0) in prokaryotes, RNA polymerase σ unit is the first
protein that assembles to the promoter (called the σ factor) recognises promoter
σ interaction with promoter with sequences located at -10 and -35 defines:
Transcriptional initiation start point, therefore σ is an initiation factor
Transcriptional direction and
Transcriptional intensity: how much of the DNA is transcribed if it is transcribed highly
dependent (if not completely) on the binding strength of σ-factor with the promoter, the
higher kd, the less RNA produced note that the equilibrium of binding reactions depends
on the concentrations of the binding partners
The promoter sequence determines the intrinsic frequency of σ complex – RNA
polymerase transcription initiation (in the absence of a repressor or activator protein)
The optimal σ-RNA polymerase promoter sequence is the consensus sequence
σ-factors also assist the RNA polymerase to separate the DNA strands at the 0 point, which is the
transcription start site, and the insertion of the coding strand into the polymerase active site
therefore transcription starts at +1
2
,Transcriptional activity varies between different genes due to the fact that:
Different σ-factors recognise different consensus promoter sequences
Presence of second messenger due to environmental clues activators/repressors binding
Environmental factors in a broad sense largely influence regulatory systems of the genes
The action of repressors binding the operator block binding of σ-factor
The action of enhancers (more dominant in eukaryotes): activator binding sites located
generally 80-160 bp upstream of transcription start site increase the binding strength
of the “special” σ54-factor with promoter
Signalling via regulatory modules
The environmental factors impose their influence on transcriptional activity via a regulatory
module: a set of a signal sensor that senses environmental clue, and an activator that carries out
its DNA binding function when activated by the sensor DNA binding at the enhancer or
operator when its binding strength is increased by the stimulus of the sensor
This module can be composed of different components:
One component regulatory module: sensory and activator domain located in one
protein, e.g. cAMP binding protein (CAP)
- Fast
- Relatively low error prone
Two component regulatory module: sensor domain separated
from activator (sensor doesn’t bind DNA), e.g. Gln level sensor;
composed of NtrB and NtrC
- Often involves kinase activity: signal amplification
- More fine tunable network flexibility (signal integration)
Note that increasing organism complexity requires a higher degree of self-organisation and therefore
more complex gene regulatory systems
Examples of regulatory modules are:
- Activators
- Repressors
RNA polymerases
Eukaryotes have RNA polymerases I, II and III, that are larger than prokaryotic ones.
RNA polymerase I: produces precursor ribosomal RNA in the nucleolus
RNA polymerase II: produces several RNAs in the nucleus
mRNA (protein encoding)
snRNA
siRNA
miRNA
RNA polymerase III: produces small RNAs in the nucleolus
tRNA
rRNA
Eukaryotic RNA polymerase II has, oppositely to I and II, a conserved CTD (carboxyl terminal domain)
that consists of n repeats of (YSPTSPS)n phosphorylation possible at several Serine positions
yields various phosphorylation patterns: corresponds to a specific transcriptional event as in each
transcriptional phase, different kinases are active: Dynamic phosphorylation and dephosphorylation
throughout the transcriptional cycle
phosphorylation pattern determines the binding position of the RNA polymerase
RNA polymerase II Ser 2: localisation to the coding region
RNA polymerase II Ser 5: localisation to the promoter region
3
, . Phosphorylation patterns are recognised by various reader proteins: enzyme complexes involved
in numerous transcription-coupled processes (e.g. capping enzymes)
visualisation with chromatin immunoprecipitation of RNA polymerase II that allows for
fractionation
The amount of CTD-p is an indicator of the transcriptional activity easily visualised in Drosophila
polytene chromosomes: ~1000 parallel DNA strands enable powerful rhodamine and fluorescent
staining with anti-phospho-CTD-rhodamine and anti-unphosphorylated-CTD-fluorescein
Key concepts lodish section 9.1
Gene expression in both prokaryotes and eukaryotes is regulated primarily by mechanisms
that control gene transcription
The first step in the initiation of transcription in E. Coli is the binding of a σ-factor complexed
with an RNA polymerase to a promoter
The nucleotide sequence of a promoter determines its strength, that is, how frequently
different RNA polymerase molecules can bind an initiate transcription per minute
Repressors are proteins that bind to operator sequences that overlap or lie adjacent to
promoters. Binding of a repressor to an operator inhibits transcription initiation or
elongation
The DNA-binding activity of most bacterial repressors is modulated by small-molecule
ligands. This allows bacterial cells to regulate transcription of specific genes in response to
changes in the concentration of various nutrients in the environment and metabolites in the
cytoplasm
The lac operon and some other bacterial genes are also regulated by activator proteins that
bid next to a promoter and increase the frequency of transcription initiation by interacting
directly with RNA polymerase bound to that promoter
The major σ-factor in E. Coli is σ70, but several other, less abundant σ-factors are also found,
each recognizing different consensus promoter sequences or interacting with different
activators
Transcription initiation by all E. Coli RNA polymerases, except those containing σ54, can be
regulated by repressors and activators that bind near the transcription start site
Genes transcribed by σ54-RNA polymerase are regulated by activators that bind to enhancers
located about 100 bp upstream of the start site. When the activator and σ 54-RNA polymerase
interact, the DNA between their binding sites forms a loop
In two-component regulatory systems, one protein acts as a sensor, monitoring the level of
nutrients or other components in the environment. Under appropriate conditions, the γ-
phosphate of an ATP is transferred first to a histidine in the sensor protein and then to an
aspartic acid in a second protein, the response regulator. The phosphorylated response
regulator then performs a specific function in response to the stimulus, such as binding to
DNA regulatory sequences, thereby stimulating or repressing transcription of specific genes
Transcription in bacteria can also be regulated by control of transcriptional elongation in the
promoter-proximal region. This control can be exerted by ribosome binding to the nascent
mRNA, as in the case of the E. Coli trp operon, or by riboswitches, RNA sequences that bind
small molecules, to determine whether a stem-loop followed by a string of uracils forms,
causing the bacterial RNA polymerase to pause and terminate transcription
4
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