Lecture 1 and 2 Flashcards
3 primary branches of the Tree of Life
Bacteria, Archae and Eukaryotes
Features of prokaryotic organisms
① Eubacteria and Archaea
② Single called
③ Lack nucleus and organelles
Features of eukaryotic organisms
① Plants, fungi, animals and humans
② Single celled or multicellular
③ Have nuclei or organelles
What does our microbiodata include?
Bacteria, archaea, fungi, protests and viruses
Microbial cell: human cell ratio
1:1
Microbioma
Combined genome of nicrobiota
Genome
It encodes the information to construct and maintain an organism.
What are genomes made of?
Most are made of DNA but some viruses have RNA genomes.
The first product of genome expression
Transcriptome
What is the transcriptome?
All the RNA present in a cell at a particular time
What is used to analyze the transcriptome?
DNA microarray
DNA microarray
It gives a snapshot of the transcriptome. It analyses RNA, not DNA!!!
How is the DNA microarray read?
It is read as a table. Red - lots of RNA, Green- less RNA, and Black- medium RNA.
How is the transcriptome maintained?
Through the process of transcription. DNA to RNA.
The second product of genome expression
Proteome
Proteome
The collection of all proteins in a cell, it defines the biochemical functions of the cell.
How is the proteome analyzed?
2D Gel Electrophoresis
How is 2D Gel electrophoresis read?
As a graph
x-axis- isoelectric point (goes from acidic to basic)
y-axis- molecular weight (goes from low to high)
How is the proteome maintained?
Through the process of translation. (RNA to protein)
‘Central Dogma’ of molecular biology
Genome (DNA) to Transcriptome (RNA) to Proteome (Protein)
How do we produce different cell types?
Through differences in genome expression. At any one time only 30-60% of genes are expressed.
Why is the regulation of gene expression crucial?
- Defining cell types (multicellular organisms)
- Responses to extracellular stimuli (both multicellular and unicellular organisms)
Promoter
Region of DNA that positions RNA polymerase and indicates transcription start site.
RNA polymerase holoenzyme
Sigma factor + core enzyme
When is the sigma factor released?
Once approximately 10 nucleotides are synthesized.
How is gene expression in both prokaryotes and eukaryotes regulated?
Gene regulatory proteins (transcription factors) which bind to the regulatory regions of DNA (CIS elements)
What can Gene Regulatory proteins do?
They can turn genes on (positive regulators- activators) and off (negative regulators- repressors)
How were gene regulatory proteins discovered?
Bacterial genetics
E. coli
- Unicellular prokaryote
- It has one chromosome of circular DNA
- It encodes about 4300 proteins
- Many genes are transcriptionally regulated by food availability
Operon
A system where multiple genes can be transcribed into a single RNA molecule
The Tryptophan (Trp) Operon
It encodes enzymes for tryptophan biosynthesis.
transcription regulated by a single promoter.
contains 5 genes.
2 potential bound states of the Tryptophan operon promoter
- Bound by RNA polymerase - Trp gene expression on
- Bound by a tryptophan repressor protein - Trp gene expression off
Where does the Trp repressor bind?
On a specific DNA sequence of the promoter called an operator
Tryptophan repressor
it blocks promoter access so that RNA polymerase cannot bind and it thus negatively regulates Trp expression.
How is the tryptophan repressor DNA binding activity regulated?
The repressor must bind two molecules of tryptophan to bind to DNA.
What is the structure of the tryptophan repressor?
It contains a helix-turn-helix DNA binding motif which binds in the major groove of the DNA double helix.
What does tryptophan binding induce?
- Conformational change
- Protein fits into the major groove
E. coli Lac operon
- Here, three genes are required for the transport of lactose into the cell and its catabolism
- it enables the use of lactose when there is high lactose and low glucose.
lac operon activator
catabolite activator protein (CAP)
it promotes lac expression: low glucose/high lactose
it binds to the CIS regulatory sequence for CAP
lac operon repressor
lac repressor protein
it inhibits lac expression: low lactose
binds to the lac operator
What does the 1st gene of lac operon encode?
It encodes beta-galactosidase; to break down lactose to glucose and galactose
How is lac operon regulated (lactose)?
- when lactose levels are low, the lac repressor is bound to the operator
- lac operon gene expression is off
Biomolecular explanation for how lac operon is regulated for lactose
increases in lactose increases levels of allolactose (related to lactose), and requires beta-galactosidase.
Allolactose then binds to the lac repressor
What happens after allolactose binds to the repressor?
- Conformational change
- DNA binding activity reduces
- The repressor is released from the operator
Why is an activator needed for RNA pol binding to lac promoter?
- RNA pol binding is inefficient to the lac promoter.
- the activator - CAP - is needed for efficient binding.
- CAP contains a helix-turn-helix binding domain
Biomolecular explanation for how lac operon is regulated for glusoce
- CAP DNA binding activity is regulated by low glucose.
- Decreasing glucose levels increases the levels of signaling molecule cyclic AMP
- cAMP will bind CAP protein, there will be a conformation change, increase in DNA binding activity and it will bind to the CAP binding site.
- RNA pol will then bind
what does a decrease in glucose cause?
increase in levels of signaling molecule cyclic AMP.
what happens after cAMP binds to the CAP protein?
- conformaitonal change
- increase in DNA binding activity
- it will bind to the CAP binding site
