Advanced molecular biology exam
Inhoudsopgave
Molecular biology techniques .......................................................................................................................... 1
Transcriptional regulation lecture 1.1 .............................................................................................................. 2
Basics of transcriptional regulation .................................................................................................................... 2
Transcriptional regulation lecture 1.2 .............................................................................................................. 5
Transcriptional regulation lecture 2.1 .............................................................................................................. 9
Transcriptional regulation lecture 2.2 ............................................................................................................ 14
Transcriptional regulation lecture 3.1 ............................................................................................................ 17
Transcriptional regulation lecture 3.2 ............................................................................................................ 20
Post-transcriptional regulation lecture 1 ........................................................................................................ 26
Post-transcriptional regulation lecture 2 ........................................................................................................ 33
Post transcriptional regulation lecture 3 ........................................................................................................ 42
Cytoplasmic regulation ..................................................................................................................................... 42
DNA metabolism – up to mismatch repair...................................................................................................... 51
The three R’s of DNA metabolism ..................................................................................................................... 51
DNA repair ........................................................................................................................................................ 55
Cell cycle 1 Lecture 2 ...................................................................................................................................... 61
Molecular biology techniques
Model organisms are highly useful in understanding human disease. Yet findings in these
models not always fully recapitulate the human situation.
Primary vs immortalized vs induced pluripotent stem cells
Primary cells
- Primary hepatocyte culture
- Limited life span (1-2 weeks)
- Difficult to genetically manipulate
HepG2 cell line
- Immortal
- Amendable to manipulate (knock out, knock in, overexpression)
- Cancerous cells with little resemblance to hepatocytes
,iPS cells differentiated to hepatocyte like cells
- iPS derived from differentiated cells can be extensively propagated
- iPS are amendable to manipulate
- iPS derived hepatocyte like cells can mimic the in vivo situation better than cell lines
2D vs 3D celcultures
Cell aggregates in an artificial matrix self-organize into tissue like structures.
Absolute vs relative measurement
Absolute: how many molecules are present per cell/samples
Relative: how much more/less molecules are present in different cells/samples
Most molecular techniques that analyze specific
gene/mRNA/proteins measure relative abundance (e.g. treated vs
untreated, wild type vs mutant)
Absolute measurement requires standards with known
quantities/ concentration (e.g. cDNA for qRT-PCR).
Accuracy vs precision
Accuracy: refers to how close a measurement is to the true or accepted value.
Precision: refers to how close measurement of the same item are to each other
Absolute measurement should be both accurate and
precise
Relative measurement does not necessarily need to be
accurate as far as the control is suffering from the same
bias/ inaccuracy
Transcriptional regulation lecture 1.1
Basics of transcriptional regulation
Transcription: The synthesis of RNA from DNA.
Genetic information flows from DNA into the end product:
proteins. This flow of information occurs through the sequential
processes of transcription (DNA to RNA) and translation (RNA to
protein). The first step is thus transcription, a rate limiting step
and therefore the prime target of regulation. When a certain
protein does not need to be produced, the best way is not to produce RNA in the first place,
as it is a waste of energy to produce RNA in the first place and then regulate the production
of proteins. The same is true within the transcription process itself. The initiation step is a
rate limiting and therefore the prime target of regulation. During initiation RNA polymerase
engages with the DNA, melts DNA near the transcription site and start transcription.
,Initiation
1. Polymerase binds to promotor sequence in duplex DNA. ‘closed
complex’
2. Polymerase melt duplex DNA near transcription start site, forming a
transcription bubble. ‘open complex’
3. Polymerase catalyzes phosphodiester linkage of two initial rNTPs
Elongation
4. Polymerase advances 3’ -> 5’ down template strand, melting duplex DNA
and adding rNTPs to growing RNA.
Termination
5. At transcription stop site, polymerase releases completed RNA and
dissociates from DNA.
How does the RNA polymerase ‘decide’ which piece of the DNA should be
transcribed?
The promotor, terminator and associated factors.
NOT THE OPEN READING FRAME!!
A sequence in front of the open reading frame is called the
promotor. It promotes transcription as this certain
nucleotide sequence is recognized by the RNA polymerase.
The open reading frame is a piece of DNA that starts with a start codon (ATG) and stop with
a stop codon (TAG, TAA, TGA). It also contains a nucleotide sequence which is transcribed
and that eventually leads to the formation of a protein.
Once the open reading frame is transcribed, the RNA polymerase needs to stop transcribing,
and this occurs via a terminator sequence.
Promotor recognition in prokaryotes
It contains 2 boxes. The -10 box and the -35 box. They are named because of their distance
to the transcriptional start. These two boxes are recognized by a sigma factor. The sigma
factor is a 3 in 1 protein.
Interaction between the sigma factor and the promotor defines
the:
- Start point
- Direction
- Intensity of transcriptional initiation
Promotor recognition in prokaryotes -> sigma factor
How different gene are expressed at different levels?
There are genes that need to be transcribed at a medium level, some a bit less, some are
complete silenced, and some should be highly transcribed. A suppressor sequence can sit on
the protein and prevent RNA polymerase to bind to it.
, Promotor recognition in prokaryotes: different sigma factors
recognize different promotor sequences.
Stationary phase genes are activated by the presence of the sigma
factor S. The expression of a different sigma factor will recreate the
RNA polymerase to a different set of promoters and in this way it
can regulate gene expression.
The lac operon
Common regulation for enzymes required for
lactose metabolism.
When there is much glucose present, there is no
need to transcribe lactose. A lac repressor is
bound to the promotor region. This blocks the
binding of RNA polymerase, and no mRNA is transcribed.
If there is glucose and lactose present, the lactose can bind
to the lac repressor which then undergoes a conformational
change and therefore it will not be able to bind to the
promotor region anymore. The RNA polymerase can then
bind to the promotor and there will be low transcription of
the lacZ.
If there is only lactose present, cAMP binds to the cap
protein. This causes a stronger binding of the RNA
polymerase to the promotor (because the promotor is not
the optimal binding site of the DNA polymerase). The lactose
binds to the lac repressor which then undergoes a
conformational change and therefore it will not be able to
bind to the promotor region anymore. The RNA polymerase
can then bind to the promotor which will be stimulated by
the cap protein which causes a high transcription rate of the
lacZ.
Sigma 54, the exception
Sigma 54 RNA polymerase interact with the activation from a distance. Sigma 54
is not directly binding next to the binding site; it is not interacting with regulatory
proteins. It is capable of reacting with proteins from a distance.
Inhoudsopgave
Molecular biology techniques .......................................................................................................................... 1
Transcriptional regulation lecture 1.1 .............................................................................................................. 2
Basics of transcriptional regulation .................................................................................................................... 2
Transcriptional regulation lecture 1.2 .............................................................................................................. 5
Transcriptional regulation lecture 2.1 .............................................................................................................. 9
Transcriptional regulation lecture 2.2 ............................................................................................................ 14
Transcriptional regulation lecture 3.1 ............................................................................................................ 17
Transcriptional regulation lecture 3.2 ............................................................................................................ 20
Post-transcriptional regulation lecture 1 ........................................................................................................ 26
Post-transcriptional regulation lecture 2 ........................................................................................................ 33
Post transcriptional regulation lecture 3 ........................................................................................................ 42
Cytoplasmic regulation ..................................................................................................................................... 42
DNA metabolism – up to mismatch repair...................................................................................................... 51
The three R’s of DNA metabolism ..................................................................................................................... 51
DNA repair ........................................................................................................................................................ 55
Cell cycle 1 Lecture 2 ...................................................................................................................................... 61
Molecular biology techniques
Model organisms are highly useful in understanding human disease. Yet findings in these
models not always fully recapitulate the human situation.
Primary vs immortalized vs induced pluripotent stem cells
Primary cells
- Primary hepatocyte culture
- Limited life span (1-2 weeks)
- Difficult to genetically manipulate
HepG2 cell line
- Immortal
- Amendable to manipulate (knock out, knock in, overexpression)
- Cancerous cells with little resemblance to hepatocytes
,iPS cells differentiated to hepatocyte like cells
- iPS derived from differentiated cells can be extensively propagated
- iPS are amendable to manipulate
- iPS derived hepatocyte like cells can mimic the in vivo situation better than cell lines
2D vs 3D celcultures
Cell aggregates in an artificial matrix self-organize into tissue like structures.
Absolute vs relative measurement
Absolute: how many molecules are present per cell/samples
Relative: how much more/less molecules are present in different cells/samples
Most molecular techniques that analyze specific
gene/mRNA/proteins measure relative abundance (e.g. treated vs
untreated, wild type vs mutant)
Absolute measurement requires standards with known
quantities/ concentration (e.g. cDNA for qRT-PCR).
Accuracy vs precision
Accuracy: refers to how close a measurement is to the true or accepted value.
Precision: refers to how close measurement of the same item are to each other
Absolute measurement should be both accurate and
precise
Relative measurement does not necessarily need to be
accurate as far as the control is suffering from the same
bias/ inaccuracy
Transcriptional regulation lecture 1.1
Basics of transcriptional regulation
Transcription: The synthesis of RNA from DNA.
Genetic information flows from DNA into the end product:
proteins. This flow of information occurs through the sequential
processes of transcription (DNA to RNA) and translation (RNA to
protein). The first step is thus transcription, a rate limiting step
and therefore the prime target of regulation. When a certain
protein does not need to be produced, the best way is not to produce RNA in the first place,
as it is a waste of energy to produce RNA in the first place and then regulate the production
of proteins. The same is true within the transcription process itself. The initiation step is a
rate limiting and therefore the prime target of regulation. During initiation RNA polymerase
engages with the DNA, melts DNA near the transcription site and start transcription.
,Initiation
1. Polymerase binds to promotor sequence in duplex DNA. ‘closed
complex’
2. Polymerase melt duplex DNA near transcription start site, forming a
transcription bubble. ‘open complex’
3. Polymerase catalyzes phosphodiester linkage of two initial rNTPs
Elongation
4. Polymerase advances 3’ -> 5’ down template strand, melting duplex DNA
and adding rNTPs to growing RNA.
Termination
5. At transcription stop site, polymerase releases completed RNA and
dissociates from DNA.
How does the RNA polymerase ‘decide’ which piece of the DNA should be
transcribed?
The promotor, terminator and associated factors.
NOT THE OPEN READING FRAME!!
A sequence in front of the open reading frame is called the
promotor. It promotes transcription as this certain
nucleotide sequence is recognized by the RNA polymerase.
The open reading frame is a piece of DNA that starts with a start codon (ATG) and stop with
a stop codon (TAG, TAA, TGA). It also contains a nucleotide sequence which is transcribed
and that eventually leads to the formation of a protein.
Once the open reading frame is transcribed, the RNA polymerase needs to stop transcribing,
and this occurs via a terminator sequence.
Promotor recognition in prokaryotes
It contains 2 boxes. The -10 box and the -35 box. They are named because of their distance
to the transcriptional start. These two boxes are recognized by a sigma factor. The sigma
factor is a 3 in 1 protein.
Interaction between the sigma factor and the promotor defines
the:
- Start point
- Direction
- Intensity of transcriptional initiation
Promotor recognition in prokaryotes -> sigma factor
How different gene are expressed at different levels?
There are genes that need to be transcribed at a medium level, some a bit less, some are
complete silenced, and some should be highly transcribed. A suppressor sequence can sit on
the protein and prevent RNA polymerase to bind to it.
, Promotor recognition in prokaryotes: different sigma factors
recognize different promotor sequences.
Stationary phase genes are activated by the presence of the sigma
factor S. The expression of a different sigma factor will recreate the
RNA polymerase to a different set of promoters and in this way it
can regulate gene expression.
The lac operon
Common regulation for enzymes required for
lactose metabolism.
When there is much glucose present, there is no
need to transcribe lactose. A lac repressor is
bound to the promotor region. This blocks the
binding of RNA polymerase, and no mRNA is transcribed.
If there is glucose and lactose present, the lactose can bind
to the lac repressor which then undergoes a conformational
change and therefore it will not be able to bind to the
promotor region anymore. The RNA polymerase can then
bind to the promotor and there will be low transcription of
the lacZ.
If there is only lactose present, cAMP binds to the cap
protein. This causes a stronger binding of the RNA
polymerase to the promotor (because the promotor is not
the optimal binding site of the DNA polymerase). The lactose
binds to the lac repressor which then undergoes a
conformational change and therefore it will not be able to
bind to the promotor region anymore. The RNA polymerase
can then bind to the promotor which will be stimulated by
the cap protein which causes a high transcription rate of the
lacZ.
Sigma 54, the exception
Sigma 54 RNA polymerase interact with the activation from a distance. Sigma 54
is not directly binding next to the binding site; it is not interacting with regulatory
proteins. It is capable of reacting with proteins from a distance.
