Lecture 4 Flashcards
what are the 3 RNAs found in prokaryotes and eukaryotes
mRNAs, tRNAs, rRNAs
eukaryotes have a lot more
how many RNA polymerases are there in prokaryotes vs eukaryotes? what are the ones in eukaryotes and what do they do
in prokaryotes there is a single type of RNA polymerase
in eukaryotes there are three different RNA polymerases for three different RNAs
–> RNA polymerase 1: transcribe rRNA genes
–> RNA polymerase 2: transcribe all protein coding genes
–> RNA polymerase 3: transcribe tRNA genes
what does transcription initiation in eukaryotes require?
- many proteins called general transcription factors
- they work by helping position the RNA polymerase at eukaryotic promoters
- these general transcription factors are required for any kind of transcription
what does the nomenclature mean for general transcription factors in eukaryotic RNA polymerase II - protein coding genes
TF = transcription factor
II = identifies the polymerase - ie. here it is RNA polymerase II
A = last letter identifies the subunit
ex. TFIID, TFIIB, TFIIA etc.
what do many eukaryotic promoters contain
a TATA box
located at the promoter to prompt transcription through the binding of RNA polymerase II and gathers the protein machinery needed for the process
promoters may have other consensus sequences
note: this is for RNA Polymerase II
what is the difference between the general transcription factors in eukaryotes and prokaryotes
eukaryotes use 5 general TF: TFIID (recognizes TATA box), TFIIB (positions RNA P at the start site), TFIIF (stabilizes), TFIIE (regulates the next TF), and TFIIH (helicase - unwinds the DNA at the transcription point)
prokaryotes need just one, sigma factor (technically part of the polymerase - a complex)
eukaryotes lack operons –> what does this mean and how does this compare to bacteria/prokaryotes
Eukaryotic genes are usually regulated one at a time, not grouped together.
In contrast, bacteria often use operons, which are clusters of genes regulated by a single on/off switch.
Eukaryotic DNA is packaged into chromatin which provides an additional mode of regulation –> what does this mean
in eukaryotes, DNA is wrapped around proteins called histones to form chromatin. This packaging controls how tightly or loosely the DNA is packed, which can affect gene expression. When DNA is tightly packed, it’s harder for the cell to access and read the genes, adding an extra layer of control over which genes are turned on or off.
what are the two categories of transcription factors:
- General Transcription factors
- required for any eukaryotic transcription
- helps initiate and continue transcription - Gene regulatory proteins
- gene expression is regulated by this
- these gene regulatory proteins bind to cis elements (regulatory regions of DNA)
- these proteins can turn genes ON (activators) or OFF (repressors)
what does a mediator do in eukaryotic transcription
- a gene regulatory protein binds to a cis element
- this gene regulatory protein is binded to a mediator, which acts as an intermediate between regulatory proteins and RNA polymerase, and thus signals the activation of TFs and transcription
how many gene regulatory proteins – activators and repressors – are used to regulate gene expression,
and how many base pairs away can these proteins effect?
what is one mechanism gene regulator proteins use to regulate gene transcription?
Around 2000 gene regulatory proteins encoded by the human genome are used to activate or repress gene expression
gene regulatory proteins can act over very large distances, sometimes >10,000 base pairs away
they use DNA looping:
- process in which DNA loops around to connect the TFs on cis elements to a mediator, which is then connected to the RNA polymerase II and transcription site.
- this helps sections far away on the DNA strand interact with each other and in the case of transcription regulators can either speed (activate) or slow (repress) gene expression
Eukaryotic gene regulatory proteins often function as protein complexes
on DNA
knowing this what are the functions of coactivators and corepressors?
coactivators and corepressors bind to the activator/repressor gene regulatory proteins that bind to the cis elements on the DNA
they do NOT bind to the DNA directly
for instance, a coactivator will bind to activator gene regulatory protein which is bound to the DNA.
this multiple protein binding group is known as a protein complex
how do eukaryotic activator proteins contribute to a modular design (2 ways)
eukaryotic activator proteins have:
1. DNA binding domain DBD
–> recognizes the specific cis regulatory DNA sequence
–>bottom part of the protein
- Activation Domain AD
–> accelerates the frequency/rate of transcription
—> top part of the protein
note: can mix match DBDs and ADs using biotech or through evolutionary mutation
–> if the DBD is switched out for protein 2 and the AD is for protein 1, the activator can only bind to the cis regulatory gene for protein 2, since that is the part that actually binds to the DNA.
–> even though the AD is for protein 1, it can still activate transcription by sending a signal to the TATA box.
how do activator proteins (gene regulatory activator proteins) activate transcription?
they attract, position, and modify:
- general transcription factors
- mediator
- RNA polymerase II
they can do this either:
1. directly by acting on these components
2. indirectly modifying chromatin structure
how do activator proteins directly activate transcription?
Activator proteins can bind directly to transcriptional machinery or the
mediator and attract them to promoters (like prokaryotic activators)
–> same idea as DNA looping when coactivators bind to the mediator
what are the parts of a chromatin?
Chromatin includes:
- Nucleosomes that are linked together in a “beads on a string” fashion
- nucleosomes (approx. 200 base pairs of DNA) consist of:
–> DNA (approx 140 base pairs long) wrapped around a histone octamer
–> histones include 8 proteins: (H2A, H2B, H3, and H4) x 2
–> linker DNA (10-80 base pairs long) linking the nucleosomes together
–> H1 protein before every nucleosome: acts as a paper clip which attaches the nucleosome on the DNA
what are the two models nucleosomes pack their chromatin fibers
what is the problem with this and how can it be solved
two models:
- zigzag model
- solenoid model
–> both are tightly packed models of chromatins
problem
- Transcriptional machinery (like general transcription factors and RNA Polymerase II) cannot assemble on promoters that are tightly packed in chromatin (either zigzag or solenoid model)
solution
- Activator proteins!
- they can alter chromatin structure and increase promoter accessibility
how do activator proteins indirectly activate transcription?
4 ways - state the complex used for each method
Activator proteins can alter chromatin structure
4 ways to do this
- 3 involve a chromatin remodeling complex
- 1 requires a histone modifying enzyme
Method 1. indirect method for transcription using activators
Nucleosome Sliding
- Nucleosome structure can be altered by chromatin remodeling complexes in an ATP-dependent manner to increase promoter accessibility
- ATP dependent chromatin remodeling complex binds to the nucleosome and through the use of ATP, the DNA is rolled around so the promoter is exposed for activation of transcription
Method 2. indirect method for transcription using activators
Activator Proteins and Histone chaperones
- Free DNA
method 2:
- ATP dependent chromatin remodeling complex binds to the nucleosome
- with ATP and nucleosome chaperons it removes the histone core, leaving the free DNA which will increase accessibility to the promoter for transcription
optional
- sometimes the histone is replaced with another nucleosome core and that helps it bind more loosely allowing access for transcription
Method 3. indirect method for transcription using activators
Activator Proteins and Histone chaperones
- replacing
method 3
- ATP dependent chromatin remodeling complex binds to the nucleosome
- with ATP and nucleosome chaperons, it replaces some of the proteins in the histone octamer with variant H2A-H2B dimers so the complex is bound more loosely allowing general TFs to bind
Method 4: activator proteins and histone modifying enzymes
what are histone modifying enzymes and what do you produce when you modify histones?
what are three types of histone modifying enzymes and what does each enzyme perform?
method 4
- Histone modifying enzymes produce specific patterns of histone modifications.
- when you modify histones you produce a “histone code”
three types of histone modifying enzymes
1. Kinases (phosphorylation)
2. acetyltransferase (acetylation)
3. methyltransferase (methylation)
note not all kinases etc. are histone modifying enzymes
where and how do histone modifications occur? what is a nickname of histone modifying enzymes and the proteins that work in conjunction?
histone modifications occur on the N terminus of specific amino acids on the histone tails
- histone tails: each protein in the histone has a tail that sticks out of the core. Usually the N terminus sticks out but sometimes the C terminus can also stick out and be modified
- these modifications occur via histone modifying enzymes AKA writers
“reader” proteins come in and recognize specific modifications and provide meaning to the code, ie. if the reader protein detects lets say a phosphorylated amino acid they may signal gene expression or silencing
