Exam 2 Flashcards
What are the 3 RNA polymerases found in all eukaryotic nuclei?
RNAPI, RNAPII, RNAPIII
Describe RNAPI (what it is, what genes it transcribes)
- Has a cleft that can encloses DNA
- Multi-subunit enzyme
- rRNA
rRNA is responsible for ______ of total cellular transcription
1/2
What transcription factor complexes are involved in the assembly of the Pol II basal transcription
complex?
TFIIA, TFIID, TFIIB, TFIIF, TFIIE/TFIIH
What are the characteristics of enhancer elements? These characteristics can help define whether a DNA element is an enhancer.
- Distant regulatory sequences that can increase gene expression
- Lack promoter activity
- Unable to direct transcription themselves
- Works in either orientation and from upstream or downstream
- Frequently have tissue specific activity
What is a silencer? How can a silencer inhibit gene expression?
- Can act on distant promoters to inhibit gene expression
- Can inhibit gene expression at the chromatin level by recruiting factors that direct the tight packing of adjacent DNA OR by binding a protein that then directly inhibits gene expression by interacting with RNA pol and its associated factors
What are homeotic genes and what is their importance/how can their gene expression be regulated?
- Genes that influence body patterning
- They encode proteins that can bind DNA
- Mutations in homeotic genes block normal development
- Can be regulated by inactivation/improper activation of TF’s
What can activators interact with during transcription?
- TAFs in the TFIID complex
- Mediator complexes
- Co-activators
- Modulators of chromatin structure such as chromatin remodeling complexes or HATS
- Components of TFIIB, TFIIE, TFIIH
How is RNA polymerase pausing overcome? What factors are involved and what is the site of action?
- A second phosphorylation event
- pTEF-b is recruited and Ser2 in carboxy terminus of RPB1 is phosphorylated
What functions do polyadenylation signals have? Or what role(s) do they serve? What can happen if an RNA de-adenylation enzyme removes a poly(A) tail from a transcript?
- Direct internal cleavage of RNA downstream of AAUAA
- Involved in termination
- The de-capping complex removes the 5′ cap, leading to a degradation of the RNA.
There are two genes that are coregulated and in close proximity, but are on separate chromosomes. Describe a method to determine chromosome conformation.
Use the 3C technique
- Cross-link with formaldehyde
- Purify DNA
- Cut DNA with restriction enzyme
- Ligate DNA
- Analyze DNA to identify segments that associate in cells
Describe a method used to show a transcription factor is binding to a particular DNA sequence.
Could use either DNA mobility shift assay, DNaseI Footprinting assay or chromosome immunoprecipitation
RPB1 is post-translationally modified on what domain? What residues? By what enzymes? What is the role of these modifications in transcription? These modifications have multiple functions. Can you name the multiple functions?
- C-terminal domain
- 5th and 2nd serine residue
- Enzyme: TFIIH (5th) and pTEF-b (2nd)
- Permits initiation of transcription and releases pausing (2nd serine residue), permitting elongation
What groove in the DNA helix does the recognition helix bind to?
The major groove
By what mechanisms can hetero-dimerization influence gene expression?
- Half sites remain similar, but the spacing between the half sites changes
- Allows for flexibility in the type of recognition element
- Change affinity for DNA
- Change specificity for DNA sites (due to different spacing)
- Lead to inactivation of transcription (think MyoD complexing with ID, which has a dimerization domain but not a DNA BD)
What is the importance of 5’ capping of mRNA?
It protects the RNA from degradation, recruits complexes involved in RNA processing, export and translation initiation, and marks cellular mRNA as “self” to avoid recognition by the innate immune system
Can you explain what synergistic activation is?
When activators work together to result in a readout much greater than what either could do alone
What are the function of ESEs?
- ESE = exon splicing enhancers
- Needed for proper splicing
What type of repeats can transcription factors bind to in DNA?
Direct or palindromic repeats
Why is it beneficial to couple processes such as transcription, RNA capping, RNA splicing, and polyadenylation?
Ensures correct processing of RNA
Can you list some functions of SR proteins?
- Recruit snRNPs to RNA to be spliced
- Help to determine which splice sites are joined together
What ensures that mRNA travels through nuclear pore complex 5’ end first? Explain how this is accomplished
- TREX
- CBC recruits TREX
- TREX recruits REC to 5’ end
- RECs promote transport to cytoplasm
What complexes or factors control RNA transport through the nuclear pore into the cytoplasm? What do these factors interact with?
- SRs bind ESEs
- SR dephosphorylation recruits RECs
- RECs promote transport to cytoplasm
OR
- H2B-Ubi promotes Ubi of CPSF protein
- Ubi-CPSF recruits REC
- RECs promote transport to cytoplasm
OR
- CBC recruits TREX
- TREX recruits REC to 5’ end
- RECs promote transport to cytoplasm
What is the wobble effect of tRNA? Can you give an example of codon sequences with variation in the wobble position?
- The wobble effect allows one tRNA to recognize more than one codon; allows different codons to direct incorporation of same amino acid
- Example: U at 3rd position (wobble position) binds
normally to A but also to G
What types of genes are transcribed by the three different RNA polymerases?
- I: ribosomal RNA
- II: all protein-coding genes
- III: transfer RNA, 5S ribosomal
Define/describe small nuclear RNAs
- 56-217 bases in size, uridine rich
- Localized in nucleus
- Important for splicing
Define/describe RNA splicing
A post transcriptional process by which introns and intervening sequences are removed from the RNA transcript
Define/describe transfer RNAs
A type of RNA molecule that helps decode a messenger RNA (mRNA) sequence into a protein
Define/describe rRNA
Molecule in cells that forms part of the protein-synthesizing organelle known as a ribosome and that is exported to the cytoplasm to help translate the information in messenger RNA (mRNA) into protein
Define/describe spliceosome
- A complex structure that consists of 5 RNAs and over 100 proteins
- Catalyzes splicing
Define/describe RNA polymerase
Enzymes that can copy the DNA into RNA together with a variety of TFs that can stimulate or inhibit polymerase activity
Define/describe holoenzyme
- A complete, functional enzyme that is catalytically active
- Apoenzyme + cofactors
Define/describe TAFs
- TBP-associated factors
- forms the TFIID complex with TBP
Define/describe nucleolus
- Found within the cell’s nucleus
- Contains several hundred copies of rRNA genes
- Has proteins involved in transcription of rRNA genes by Pol I and proteins involved in the processing of 4S RNA into 28S, 18S and 5.8S rRNAs
Define/describe p-TEFb
- A kinase that is recruited and phosphorylates RNAP II so that transcription continues and a full RNA transcript is produced
- Required for transcriptional elongation
Define/describe polymerase pausing
A mechanism that cells use to stop and synchronize transcription
- Phosphorylation of Ser5 by TFIIH permits initiation of transcription
- Recruitment of kinase pTEF-b
- Phosphorylation of Ser2 permits elongation of transcription
Define/describe polyadenylation signal
AAUAA
Define/describe core promoter
- A region of DNA close to transcriptional start site which directs transcription
- Sequences adjacent to the transcriptional start site that control the expression of a gene
Define/describe upstream promoter elements
- From -300 to -100
- Upstream/before core promoter
- May serve as targets for transcription factor binding or may interact directly with the core transcriptional machinery or both
Define/describe DNA mobility shift assay
- Radioactively label DNA fragment
- Mix with whole cell or nuclear extract and incubate
- Run the mixture on a nondenaturing electrophoresis gel and observe the position of the radioactive bands by autoradiography
- Detect protein binding to the DNA by the appearance of a shifted band
Define/describe DNase1 foot-printing assay
- Radioactively label DNA fragment or oligonucleotide with sequence of interest at one end only
- Mix with whole cell/nuclear extract and incubate
- Briefly digest with DNase1, producing a series of fragments of lengths differing by one nucleotide
- Run on a denaturing gel and detect radioactive bands by autoradiography
- Areas with gaps indicate the presence of a protein
Define/describe silencers
A regulatory DNA sequence that can decrease the rate of transcription of a structural gene
Define/describe DNA binding domain
An independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA
Define/describe expression vector
Usually a plasmid or virus designed for gene expression in cells, used to introduce a gene/TF to a target cell
Define/describe homeodomain
- A DNA binding domain made up of 60 amino acids
- contains a helix-turn-helix motif
Define/describe recognition helix
A helix within the helix-turn-helix that mediates sequence specific binding and binds to the major groove
Define/describe co-repressor
A protein that does not itself bind DNA but assembles on other DNA-bound gene regulatory proteins to inhibit the expression of a gene. Can mediate the effect of DNA-bound repressors
Define/describe zinc-finger
- 30 amino acid repeating sequence
- 2-cysteine 2-histidine
- found in many TF’s
- zinc finger is anchored at its base with conserved cysteine and histidine residues that coordinate zinc
- consists of 2 antiparallel beta sheets and an alpha helix
Define/describe beta-sheets
Important for coordinating zinc
Define/describe alpha-helix
- Important for recognizing DNA
- Contacts DNA major groove
Define/describe homodimer
A protein composed of two polypeptide chains that are identical in the order, number, and kind of their amino acid residues
Define/describe leucine zipper
- A dimerization domain that allows DNA binding by the adjacent basic domain
- Does not bind directly to DNA
Define/describe helix-loop-helix
- Does bind directly to DNA
- A motif that is found in a number of transcription factors which regulate gene expression during embryonic development
- DNA binding domain when found in homeodomains
Define/describe MyoD
A TF/gene that’s ability to stimulate gene expression is inhibited by heterodimerization with the Id factor, which has a helix-turn-helix domain but no basic domain
Define/describe domain-swapping
A technique with the goal of identifying activation domains
- Combine DNA-binding domain of 1 with different regions of 2
- Test on gene carrying binding site for Factor 1
- Look for gene activation
Define/describe basal transcription complex
- Considered to be the basic set of proteins needed for transcription in eukaryotes
- Consists of RNA Pol II, TFIIA, D, B, F, E, and H
- First TFIIA and TFIID bind to the promoter, followed by TFIIB, TFIIF/RNA Pol II and TFIIE/TFIIH
Define/describe a mediator
A multiprotein complex that functions as a transcriptional coactivator in all eukaryotes
Define/describe HIV, HI
Viral Tat protein
- Binds viral RNA
- Promotes Pol II initiation
- Overcomes pause
- Enhances elongation
Define/describe a super elongation complex (SEC)
- Multiprotein complex that contains pTEF-b
- Is required for the proper induction of the HSP70 gene upon stress, and this complex is also involved in HIV proviral transcription
- Plays a key role in stimulating transcriptional elongation of cellular as well as viral genes
Define/describe polycistronic
Containing the genetic information of a number of cistrons; encodes more than one protein
Define/describe monocistronic
- A type of messenger RNA that can encode only one polypeptide per RNA molecule
- mRNA in eukaryotes are typically monocistronic
Define/describe Shine-Dalgarno sequence
- Ribosomal binding site in bacterial messenger RNA
- It enables initiation of protein synthesis by aligning the ribosome with the start codon
Define/describe CPSF
The central component of the 3′ processing machinery for polyadenylated mRNAs: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage
Define/describe cap-binding complex
Binds to the capped RNA in the nucleus, protects it from degradation and facilitates its transport to the cytoplasm
Define/describe poly (A) polymerase
The enzyme that adds a chain of adenines (poly A tail) to the 3’ end of an RNA transcript, making the RNA more stable and protecting it from degradation
Define/describe lariat
A lasso‐shaped structure formed from the fragment of RNA to be excised during the process of splicing out the introns in the primary transcript of eukaryotic genes
Define/describe branch point
The A residue where the lariat closes itself to form a loop
Define/describe snRNPs
RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs
Define/describe splice site
The junction between the upstream exon and beginning of an intron
Define/describe exon-splicing enhancers (ESE)
Facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron
Define/describe SR proteins
- S = serine, R = arginine
- Have 3 functions/roles: bind ESEs, recruit snRNPs and direct splice site joining
Define/describe CTD
- C terminal domain
- largest subunit of RNA Pol II, has been demonstrated to play important roles in capping, splicing, and 3′ processing of mRNA precursors
Define/describe exon skipping
- Occurs when there is a mutation in ESE
- Exon not bound by SR is spliced out
- Associated with human diseases
Define/describe RNA exporter complex (REC)
- Needed for a fully processed mRNA to be transported through the nuclear pore
- Involved in various post-transcriptional processes like capping, adenylation, and splicing
Define/describe TREX
Helps RNA 5’ end pass through nuclear pore first
Define/describe wobble effect
A hypothesis that describes how one amino acid can be encoded by several codons
Define/describe initiator tRNA
It reads the start codon, allowing the initiating ribosome to begin translation in the correct location
Define/describe proof reading
The process by which incorrect or mismatched bases are corrected
Define/describe exosome
-In nucleus: a complex that degrades introns and
incorrectly spliced RNAs
- In cytoplasm: complex is involved in normal turnover functional mRNAs
Define/describe nonsense mediated RNA decay
- A second RNA degradation mechanism that exists in the cytoplasm
- Recognition and degradation of nonfunctional mRNAs that are nonfunctional due to a premature stop codon
Define/describe EJC
- Complex that binds to each exon during splicing at a position 20-24 bases upstream of the junction between two exons and remains associated to it during export to the cytoplasm
- Normal location of stop codons is downstream of last EJC
What genes does RNA Pol II transcribe?
All protein-coding genes
What genes does RNA Pol III transcribe?
Transfer RNA, 5S ribosomal
Describe the association between DNA and RNA Pol I
- RNA Pol I is not recruited to DNA by direct binding
- Other protein factors bind DNA and recruit RNA Pol I via protein : protein
Describe the recruitment of RNAPI
- Upstream binding factors (UBF) binds promoter
- SL1 is recruited via protein : protein interactions with UBF
- SL1 recruits RNAPI and transcription starts
Describe RNA Pol III
- Multi-subunit enzyme
- Has a cleft that can enclose DNA
Describe the association between DNA and RNA Pol III
- Recruitment to DNA is NOT by direct binding
- Other protein factors bind DNA and recruit RNA Pol I via protein : protein
Describe the recruitment of RNAPIII
- TFIIIA binds promoter, then TFIIIC
- TFIIIC recruits TFIIIB to form a stable transcription complex
- TFIIIB recruits RNA Pol III via protein : protein interaction
Describe the recruitment of RNAPII / the assembly of the basal transcription complex
- TFIIA recruits TFIID to promoter
- TFIID binds TATA box
- TFIIB recognizes TFIID/DNA
- TFIIB binds BREs on either side of that TATA box
- TFIIB recruits RNAPII / TFIIF
- TFIIE/TFIIH recruitment forms pre-initiation complex
- TFIIH kinase activity phosphorylates RNAPII C-terminal domain of RPB-1
- Phosphorylation permits initiation of transcription
What is the TATA box?
DNA target for progressive assembly of basal pre-initiation complex of Pol II
Describe the 3D structure of TFIID
- Contains TATA binding protein (TBP)
- Binds TATA box
- DNA fits in groove
What is the role of TFIIA in the assembly of the basal transcription complex?
Recruits TFIID to the promoter
What is the role of TFIID in the assembly of the basal transcription complex?
Binds the TATA box
What is the role of TFIIB in the assembly of the basal transcription complex?
- Recognizes TFIID/DNA
- Binds BREs on either side of TATA box
- Recruits RNAPII / TFIIF
What is the role of TFIIE / TFIIH in the assembly of the basal transcription complex?
Their recruitment forms the pre-initiation complex
What is the role of TFIIH in the assembly of the basal transcription complex?
Has kinase activity, phosphorylates RNAPII C-terminal domain of RPB1
What portion of RPB1 is phosphorylated?
The 5th and 2nd serine
TFIIB is equivalent to ______ for Pol I genes
SL1
RNA Pol IV and V work together to…
inactivate chromatin and inhibit gene expression via DNA methylation, deacetylation and/or methylation of H3 K9/K27
Describe how TBP is a basic TF used by all polymerases
- Pol I: part of SL1
- Pol II: part of TFIID
- Pol III: part of TFIIIB
What are transcription factories?
- Punctuate areas in nucleus
- Genes move to these sites to be transcribed
T / F : genes located on different chromosomes can co-localize to the same transcription factories
T
What are the 3 classes of RNA Pol II distribution on genes? Which one is paused?
- Inactive gene
- Potentially active gene (paused)
- Active gene
What are the 4 stages of transcription?
- Initiation
- Pausing
- Elongation
- Termination
Define/describe co-activators
Proteins that do not themself bind DNA but assembles on other DNA-bound gene regulatory proteins to activate the expression of a gene. Can mediate the effect of DNA-bound activators
