Control of Gene Expression in Eukaryotes Flashcards
How many promoters are relevant to a single eukaryotic gene?
A single promoter corresponds to a single eukaryotic gene.
What regulates transcription in eukaryotes?
Chromatin structure
When do transcription and translation occur in prokaryotes and eukaryotes?
Prokaryotes: both transcription and trAnslation occur in a coupled way.
Eukaryotes: transcription occurs first and then translation occurs at a separate time in a separate place.
What is Chromatin?
DNA that is complexed with proteins called histones
What is the net charge of a histone?
Histones are net positively charged since DNA has an overall negative charge.
What are the 5 different types of canonical histones?
H1, H2A, H2B, H3, and H4
What is a histone core?
A histone core is an octamer of 8 histones, with two copies of each of H2A, H2B, H3 and H4
What are the intervals of DNA between nucleosomes that are not bound to histones called?
Linker DNA
What are the pairing of dimers that make up core histones?
H2A-H2B
H3-H4
What is a nucleosome?
DNA wound around a histone core at regular intervals
What are histone tails?
11-37 amino acids at the N-terminus of protein that are mostly positively charged.
What are the function of histone tails?
Interact with DNA of neighboring nucleosomes which compacts the nucleosomes.
What is the function of histone H1?
Binds where DNA enters and leaves the nucleosomes (clamps DNA)
What needs to occur in Chromatin structure in order for transcription to occur?
Chromatin organization of DNA needs to be decompacted or decondensed in order for transcriptional machinery to bind and activate transcription.
What is DNase I?
DNase I cuts phosphodiester bonds in DNA (DNA bound to histone proteins are less sensitive to DNase I).
What are DNase hypersensitive sites
They are typically present upstream of start site of transcription of genes and their increased sensitivity of being cut by DNase I
What are the three mechanisms used to alter Chromatin structure?
Chemical modification of Histones
Chromatin remodeling
DNA methylation
What are the two histone domains and what are their function?
C terminal domain: protein fold that interacts with phosphates of DNA as well as other histones in the same nucleosome.
N terminal domain: an overall positively charged tail that interacts with phosphates of DNA of the same nucleosome and with DNA of neighboring nucleosomes.
What are the histone modifying enzymes correlating to phosphorylation?
Phosphorylation (kinases)
Dephosphorylation (phosphatases)
What are the histone modifying enzymes that are associated with methylation?
Methylation (methylases)
Demethylation ( demethylases)
What are the histone modifying enzymes that are associated with acetylation?
Acetylation ( acetyl transferase)
Deacetylation ( deacetylases)
What does phosphorylating or acetylating a histone generally do?
Usually enhances transcription by weakening interaction of histone with DNA by introducing a negative charge on amino acids
What does methylation of histones do?
Can result in either activation or repressor of transcription depending on what particular amino acid in histone tail is modified and the number of methyl groups attached to amino acid.
What are Chromatin remodeling complexes and what are their modes of action?
Reposition of nucleosomes: slide nucleosomes along DNA or eject nucleosomes
Replace canonical histones in nucleosomes with histone variants that have slightly different amino acid composition in comparison to canonical histones
What do Chromatin remodeling complexes need in order to function?
ATP
How are Chromatin remodeling complexes targeted to DNA sequences? (2 ways)
Binding transcriptional activators or repressor bound to specific DNA sequences
Binding pre-existing histone chemical modifications.
What needs to occur for DNA methylation to repress transcription?
Methylation of cytosines in CpG dinucleotides near transcription start sites of genes. There are usually several CpG dinucleotides clustered together near the transcription start site of genes.
What is a CpG island and where do they typically reside?
CpG islands are several CpG dinucleotides clustered together that are near transcription start sites of genes.
What do methylated CpGs recruit?
Protein complexes containing histone deacetylases which increase Chromatin compaction therefore repressing transcription
What is an epigenetic phenomena
Alterations to Chromatin structure that affect gene expression and that do not involve changes in nucleotide sequence of DNA.
What do transcriptional regulatory proteins bind to?
Specific sequences in regulatory promoters and enhancers/silencers.
How do coactivators function?
Coactivators do not directly bind to DNA but through binding to activities they enhance the transcriptional activity of activators
How do transcriptional activators function?
Through binding to regulatory enhancers/promoter and interact with basal transcription apparatus directly or via mediator to enhance stable binding of basal transcription apparatus to the core promoter this increasing the rate of initiation of transcription.
What is the basal transcription apparatus?
The basal transcription apparatus binds to core promoter and includes general transcription factors that are involved in the initiation of transcription.
In which ways can general transcription factors and transcriptional regulatory proteins function?
Intrinsic histone-modifying enzymatic activity
Recruit histone-modifying exhumes and Chromatin remodeling complexes
What is Gal4?
Gal4 is a transcriptional activator that binds an enhancer, UAS (upstream activating sequence for Gal4) present in genes that encode gene products required for galactose metabolism in yeast.
How is Gal4 blocked from activating transcription?
Gal80 blocks Gal4 from activating transcription when galactose is absent.
How does Gal3 affect Gal80 in the presence of galactose?
Gal3 binds to Gal80 causing a conformational change in Gal80 releasing Gal80 from Gal4 which allows Gal4 to interact with the basal transcription apparatus thus stimulating transcription.
How do transcriptional repressor work in eukaryotes?
Bind to consensus sequences in regulatory promotors or silencers and repress transcription
How do Transcriptional repressors function?
Compete with transcription activators for the same or overlapping DNA binding sites
Bind to their binding sites in DNA near transcriptional activator binding site and by interacting with the activator, prevents activator from contacting basal transcription apparatus
Directly interfere with stable assembly of basal transcription apparatus at core promoter
What are enhancers?
Sequences that can be present at a distance from a core promoter and function in a position and orientation independent manner to activate transcription
How do transcriptional activators use enhancers?
Transcriptional activator proteins bound to enhancers contact basal transcription machinery bound to the core promoter, stabilizing the binding of basal transcription machinery to promoter.
What are enhancer RNAs (eRNAs)?
Enhancer sequences transcribed into an RNA sequence that does not get translated. Transcription of eRNAs is associated with transcription at promoters regulated by the enhancer.
What is a super-enhancer and what does it do?
A super enhancer is several enhancers clustered together that causes high levels of transcription.
What are insulators and what do they do?
DNA sequences that block or insulate effect if enhancers via proteins bound to them.
What are TADs
Large spatially interacting regions of Chromatin seperated physically from other TADs
What do proteins that bind to insulators do?
Proteins that bind insulators create topologically associated domains (TADs) in Chromatin.
What do TADs do
TADs are Chromatin loops that allow ehpnhancers to interact with promotors within that loop but not with promotors of other TADs essentially creating neighborhoods of enhancer that physically interacts with core promotors of a gene it regulates, but this gene is insulated from enhancers of other neighborhoods.
What is coordinated gene regulation?
Genes that are coordinated regulated by the same stimulus have short regulatory sequences (response elements) in common in their enhancers/regulatory promotors
What is gene regulation through RNA splicing?
Alternative splicing allows a pre-mRNA to be spliced in multiple ways which generates different proteins in different tissues or at different times during embryonic development.
What is gene regulation through RNA degradation?
Genes can be regulated by the degradation of RNA sequences stopping translation through ribonucleases (RNAses). The steady state level of mRNA is dependent on the rate of synthesis and degradation of mRNA
What are the 3 ways RNA can be degraded by RNases?
Shortening of polyA tail leading to the removal of 5’ cap
Removal of nucleotides from the 3’ side of mRNA
Cleavage of RNA strands at internal sites
What triggers RNA interference?
Small RNA molecules 21-25 nucleotides long called siRNAs (small interfering RNAs) or miRNAs (micro RNAs)
What is RNA interference?
siRNAs and miRNAs binding target mRNA sequences within the RNA inducing silencing complex (RISC) which either cleaves a region of target mRNA bound by siRNA and then degrade their target mRNAs or inhibit their translation.
