What are the major differences between prokaryotic and eukaryotic transcription?
- Transcription and translation are in separate compartments
- Eukaryote pre-mRNAs are subject to extensive post-transcriptional modification – processing
- Chromatin structure in eukaryotes limits accessibility (transcription is highly regulated; only 0.01% of genes in a typical eukaryotic cell are undergoing transcription at any given moment)
- Eukaryotic RNAP does not recognize binding site – needs general transcription factors to help
- Mostly multicellular organisms (different cells/tissues)
- Three RNA polymerases with different roles
Which hypothesis regarding eukaryotic RNAPs was proven with α-amanitin and actinomycin D (be specific)?
- The hypothesis that different polymerases transcribe certain genes but not others was proven with the compounds α-amanitin (decreases the activity of RNA polymerase II and III) and actinomycin D (decreases the activity of RNA polymerase I)
- The compounds were injected in mouse cell nuclei and then the transcripts produced were subject to gel electrophoresis. Certain genes were proven to be transcribed while others were not…their transcriptional levels were dependent upon which polymerase was inhibited.
Which genes are transcribed by RNAP I? RNAP II? RNAP III?
- RNA polymerase I: transcribes rRNA (28S, 18S and 5.8S rRNA)
- RNA polymerase II: transcribes mRNA and snRNAs
- RNA polymerase III: transcribes tRNA, 5S rRNA, U6 snRNA, 7SL RNA and 7SK RNA
What does CTD stand for? Explain the role of CTD tail in eukaryotic gene expression?
• CTD stands for Carboxy-Terminal Domain
• It is a consensus sequence which is unique to RNA polymerase II
o Stretch of 7 amino acids (YSPTSPS) that are repeated multiple times (at least 10, specific to organism)
o 5 of 7 amino acids have –OH group, therefore hydrophilic and phosphorylatable
• It is part of the largest subunit in RNA polymerase II
• Deletions that remove more than half the repeats are often lethal
• Un-phosphorylated CTD tails initiate transcription; necessary for methyl cap addition and polyadenylation as well as splicing
• In areas of high transcription, only phosphorylated CTD tails are present
How would you define enhancers? What are their characteristics? What is the difference(s) between enhancer and upstream control element?
- Enhancers are site elements that stimulate transcription
- They are identified by sequences that stimulate initiation but are located at a considerable distance (up to 50 kb) from the start point of transcription (downstream or upstream)
- Its elements are usually in a closely packed array
- Transcription factors that recognize enhancers are called activators
- Sometimes the same element can be both an enhancer and silencer depending on the bound protein
- Enhancer elements are orientation independent
- They can be found ANYWHERE – making them different from an upstream control element
- Often targets for tissue-specific or temporal regulation
- Upstream control elements must be proximity to the promoter, while the enhancer can be anywhere and in any orientation
Explain the use of reporter genes for estimation of promoter strength.
- A reporter gene is a coding unit whose product is easily assayed. (Either enzymes or a fluorescent protein)
- It may be connected to any promoter of interest so that expression of the gene can be used to assay promoter function.
- The level of expression of measurable (easy to assay) gene product (enzyme) is proportional to the strength of the promoter. strength is proportional to amount gene product produced
Explain briefly 5’ deletion series. What kind of information do they reveal?
- By deleting/mutating a part of the promoter (starting from 5’ end 5’ deletion series) and repeat the experiment.
- By doing this, you will get information about how important the deleted parts of the promoter are.
- The resulting change in the expression of the gene product is measured.
- Areas of low or no transcription are important promoter elements
Explain the modular nature of RNAP II promoters.
- Modular means the promoters are organized on the principle of “mix and match”
- No one element is essential for (or common to) all promoters, and in any individual promoter, elements may differ in number, location, and orientation
- Many RNAP II promoters have TATA boxes and those that do not, have initiator sequences of CG islands instead
- Proximal elements are required for developmental, environmental, or tissue-specific expression
- Promoters that respond to similar conditions, but control expression of different genes, may have similar cis elements
- Enhancers may also have binding sites for different trans factors which may increase transcription initiation.
- Enhancer: sometimes composed of multiple binding sites for different trans factors; the combination leads to increased transcription initiation.
- Expression of one gene can be induced or increased or decreased or shut-off under different conditions due to the modularity of gene’s promoter.
Draw a diagram of RNAP II promoter (show all types of elements it could have).
• Core promoter: minimal set of elements required for accurate in vitro transcription initiation by RNAP II; necessary for recruitment, binding, and proper positioning of RNAP II
o TATA = TATA box (at ~ -30)
o BRE = TF II B recognition element
o Inr = Initiator (on the transcription start site)
o DPE = Downstream element (downstream)
• Lots of promoters do not have initiator and downstream element
• There are also TATA-less promoters
• Promoters are controlled by regulatory elements: enhancers, silencers, and upstream elements
https://68.media.tumblr.com/c025484e80b71ea8b27d3deec8b8eb6e/tumblr_otyrwwSXH51qhvmqho1_540.jpg
Explain the tissue (cell type) specificity of eukaryotic cis elements.
- The very same cis elements (including enhancers) from the promoter region are present in each tissue.
- Tissue specificity is due to either presence or absence of a particular binding protein(s)
- Binding proteins are tissue specific.
- Binding proteins (TFs) dictate tissue specificity of cis elements at the level of initiation of transcription.
Knowing that different genes may have the same promoter and enhancer elements and that different transcription factors contain the same structural features, how would you explain transcriptional specificity?
- The absence or presence of binding proteins is particular for the promoter and enhancer elements.
- A gene that responds to multiple conditions will have multiple cis elements.
If you know the binding site for certain transcription factor (TF), outline the first step you’d take.
Isolate nuclear proteins
If you know the binding site for a certain transcription factor, outline the 2nd step you’d take, and the experiments involved.
- Purify transcription factors:
a) Ion Exchange Chromatography
o Increasing salt conc. will result in different proteins binding with different affinity
o Highest salt concentration – possible highly purified TF
b) DNA-affinity chromatography
o Beads with attached DNA strands containing TF binding sites; collect fractions
c) Specific DNA-affinity chromatography
o Proteins eluted by DNA affinity chromatography subjected to DNase footprinting assay (to check to purity)
If you know the binding site for a certain transcription factor, outline the 3rd step you’d take, and the experiments involved.
- TF Activity Assay:
a) The partial amino acid sequence of the extracted TF is determined; screen library or search data bases; gene (cDNA for TF)
b) Construct 2 plasmids from the cDNA library of the extracted TF:
i. With gene encoding TF (=gene for X)
ii. With reporter gene and binding site for TF (TF is product of gene X)
c) Transfect the host cell without functional X with these 2 plasmids
d) Production of reporter gene is measured – controlled by binding of TF X
e) Could introduce mutations in the X gene or in the X binding sequence- information on important domains of the TF
Distinguish between the function of promoters and enhancers in transcriptional regulation.
• A promoter is a DNA sequence which, in eukaryotes, RNA polymerase binds to initiate transcription with the help of general transcription factors
• An Enhancer is a cis-acting sequence which increases the utilization of some eukaryotic promoters
o Can function in any orientation and in any location (upstream or downstream) relative to the promoter
Distinguish between the function of general transcription factors and transcription activators in transcriptional regulation.
- General transcription factors are necessary for the binding of RNAP II to the core promoter
- Transcription activators are factors that bind to regulatory elements to activate transcription
- ALL ARE STILL CALLED TRANSCRIPTION FACTORS
List and briefly explain four major domains in eukaryotic transcription factors.
• DNA-binding domains (DBD):
Interacts with specific DNA sequences
• Transcription activation domain (AD):
Interacts with other proteins to stimulate transcription from a nearby promoter
• Dimerization domains (homo- or hetero-dimers):
• Ligand-binding domains (LBD):
Binding of accessory small molecule regulates transcription factor activity
List most frequent structural motifs in eukaryotic DNA binding domains.
- Zinc Finger
- Homeodomain Proteins
- Leucine Zipper Proteins
- Helix-loop-helix
List three classes of transcription activation domains in eukaryotic transcription factors.
- Acidic Activation Domains
- Glutamine-rich Domains
- Proline-Rich Domains
What is achieved by the ability of some transcription factors to form heterodimers (basically two things/players in regulation)?
- Heterodimers allow for combinatorial control and can increase the DNA binding specificity to the trans-factor
- Two monomers and two different activation sites brought together to form a third and completely different activation domain
- Allows for greater complexity and diversity
What is meant by the independence of the DNA-binding and transcription-activating domain of a transcription factor?
- DNA-binding domain has no influence on the transcription activation domain and vice versa
- The activation domain is important only for activation and the DNA binding domain is important only for binding
- One does not influence the other
What is the role of the TATA box? What happens when TATA box is removed from the RNAP II promoter?
• The TATA box is a highly conserved region which has several functions:
i) Locates the start site of transcription (about 30 bp downstream)
ii) Sometimes important for the efficiency of transcription
iii) The binding protein which binds to the TATA box initiates the assembly of the general transcription factors and RNA polymerase
• The TATA box isn’t completely necessary for transcription; if it is not present, the gene must have either an initiator (core element) or GC boxes (upstream elements)
• Deletions of the TATA box lead to the shifting of the transcription start site downstream (i.e., transcription is always 30 bp downstream)
What is combinatorial control of transcription?
• Gene is only expressed when only a specific combination of proteins is present; binding of certain proteins will enable the assembly of the other proteins necessary for transcription initiation
What is the role of TFIIE and TFIIH in transcription initiation by RNAP II?
- TFIIE is DNA-dependent ATPase; binds to the already formed TFIIF/RNAIIP and helps position the polymerase over the start site of transcription and probably helps generate the energy needed to begin transcription; attracts and regulates TFIIH
- TFIIH has helicase activity, and so when bound to the complex, it helps unwind the DNA which is necessary for promoter clearance; it also phosphorylates the CTD tail of the RNAIIP which detaches RNAPII from TFIID, beginning transcription by RNAPII
