Section 4 - Regulation of Gene Expression Flashcards
(33 cards)
Describe the components needed for transcription in bacterial cells
Basically everything that’s in a bacterial Operon:
- Start Site: called +1. In order for RNA Pol to start, it must recognize the start of the gene.
- Promoter: the DNA sequence (Around 100 bp) required to initiate transcription of a gene of operon. The sigma subunit of RNA Pol recognizes promoter, and holoenzyme latches firmly onto this specific sequence of nucleotides. Lies immediately upstream (-1) of start site.
- has -10 and -35 sequences
- the polarity of the promoter orients the polymerase and determines which strand is transcribed (the one that is 3’to5’ so that it can transcribe 5’-3’)
- not transcribed into RNA
- Repressor/Terminator: the DNA sequence required to stop transcription. Transcribed into RNA.
- RNA Polymerase: The multi-subunit enzyme that makes specific RNA transcripts using DNA as a template and nucleoside triphosphates (NTPs) as substrates.
- Moves stepwise 3’-5’ along the DNA, unwinding it as it goes. Adds ribonucleotides one by one to the RNA chain. Catalyzes the formation of phosphodiester bonds that link the nucleotides + the backbone. The two strands of DNA come right back together. No primer needed.
Describe mechanisms for transcriptional activation and repression in bacteria. (Trp Operon)
- increased transcription by enhancing activity of RNA Pol or enhancing recruitment to the site Trp operon (expression of Trp) negatively regulated by Trp repressor - Little/no Trp = transcription (of the 5 genes responsible for Trp biosynthesis, aka. Trp operon) can occur. -When aa tryptophan is high in concentration, 2 Tryptophans bind trp repressor--> undergoes a conformational change now can bind to operator(btwn-10 and-35) impeding recruitment of RNA Pol --> no transcription. Trp repressor binds DNA as a dimer --- helix 5 on each of the two monomers of repressor makes BASE SPECIFIC contacts in consecutive major grooves of the DNA.
Explain Trp Operon and how it is regulated
When is it expressed?
What does the operon encode for?
Explain Lac Operon and how it is regulated
When is it expressed?
What does the operon encode for?
What does Catabolite Repression have to do with it?
-you shut off the genes required to metabolize other carbon sources in the presence of glucose. so lac operon is subject to this.
(Glucose, CAP, cAMP)
What is the role of CAP?
Two ways to regulate transcription
- Stimulate/Repress recruitment of RNA Pol
- Stimulate/Repress activity of RNA Pol
then activation is through protein-protein interactions (CAP to RNA Pol)
Identify the structural characteristics of some bacterial DNA-binding proteins
- Trp repressor
- 107 residues. a monomer
- 6 helices + some unstructured loops. Alpha helices 4 and 5 make up the helix-turn-helix motif.
- acts as a dimer – 2-fold symmetry. 2 monomers held together by protein-protein interactions. so actually needs TWO tryptophans to bind
- at either end (the heads), its helices are pointed inward (blocking interaction with operator. When 2 Trps binds to it, –> conformational change: the two recognition helix 5s on each of the two monomers tilt back out to make BASE SPECIFIC contact in consecutive major grooves of the DNA. highly specific recognition
Compare and contrast prokaryotic and eukaryotic transcription
- Euks have 3 diff RNA Pols, Proks have 1
Pol 1 – rRNA (RNA)
Pol 2 – mRNA
Pol 3 – tRNA , snRNA (5s rRNA) - Euks no operon (genes transcribed as single units – monocistronic)
- Promoter Structure
- Euk: no sigma factor/-10-35 sequences. RNA needs Promoter recognition help from/by transcription factors to start transcription. Namely TFIID, which has TATA-binding protein (TBP) attached. TBP recognizes and binds TATA box (a DNA sequence, promoter element) which is ~ 20 bps up from start site +1. (this makes it bend uniquely which attracts other transcription factors here, like TFIIB)
- Euk gene activation can occur thousands of bps away from +1 where RNA Pol is. The enhancer DNA site + activator protein (trans factor) are brought close to promoter +Pol by DNA looping. Held together by Mediator.
- Nucleosomes and higher order chromatin structure can regulate transcription by blocking/uncovering transcription factors –> by recruiting chromatin remodelling complexes to reposition nucleosomes allowing for TATA box to be accessed)
Or recruit histone-modifying enzymes (eg, histone acetyltransferases) to add acetyl groups to specific histones which then serve as binding sites for proteins - Combinational Control
- groups of proteins work together to determine the expression of a single gene
- whereas in prok, it’s either on or off. Euk gets a range of expression
SEE FIG. 8-12
Examples of protein-protein interactions (prok)
- CAP and RNA Pol
- holoenzyme: the sigma factor/subunit that helps RNA Pol find promoter
Describe the fundamental features of chromatin and how it regulates
transcription.
Transcription factors, regulators, and RNA Pol must get to the DNA amidst its high-order packing. Nucleosomes can inhibit initiation of transcription by physically blocking the promoter from the general trans factors or RNA Pol.
-Chromatin remodelling complexes
Describe the fundamental features of chromatin and how it regulates
transcription.
Transcription factors, regulators, and RNA Pol must get to the DNA amidst its high-order packing. Nucleosomes can inhibit initiation of transcription by physically blocking the promoter from the general trans factors or RNA Pol.
-Chromatin remodelling complexes: use ATP energy to loosen the DNA and push it along the histone octamer, exposing the DNA
-Reversible Chemical Covalent modifications of the histone proteins: eg. acetyl, methyl and phosphate groups can be added to/removed from the tails by enzymes that are in nucleus. e.g. recruitment of acetyl transferases promote attachment of acetyl groups to selected lysines in the tail of histones – alters/loosens chromatin structure, allowing accessibility + groups attract proteins that promote transcription (ie, trans factors)
Thus can also attract histone deacetylases, thereby reversing pos effects
- These modifications also act as docking sites for regulatory proteins
Outline mechanisms that a eukaryotic cell could use to regulate gene expression
Using example of regulating genes responsible for galactose metabolism in yeast
Positive Reg: Yes Galactose = Yes transcription
-
Outline mechanisms that a eukaryotic cell could use to regulate gene expression
Using example of regulating transcription at estrogen receptor:
□ Has 3 domains
a) Transcription-activating domain –Calls RNA polymerase to promoter
b) DNA-binding domain in middle –Zinc finger.
c) Ligand binding domain –Binds inhibitory proteins and Ligand = estrogen
No estrogen = No transcription (bc inhib proteins)– in cytoplasm
Yes estrogen = Estrogen binds to LBD, conf change of ER, bye inhib protein, translocation to nucleus (now ACTIVE). Now ER binds to ERE (DNA sequence) on promoter. Co-activator protein binds, attracting CRCs and histone modifiers —> TRANSCRIPTION
-Can be ligand/estrogen-INdependent: environment phosphorylates ER, mimics, releases inhib protein, etc. Results in transcription of specific genes with ERE. Application in Cancer.
-Can activate non-ERE genes (ER=promiscuous): Estrogen-ER complex (or phosphorylated ER) binds to transcription factors that bring it to promoters that don’t have ERE (eg, trans factor AP1 brings it to its promoter –> increased transcription)
When do yeast express the genes required to metabolize galactose?
idk
Provide examples of where protein-protein interactions regulate transcription. (euk)
Spliceosome holding the snRNPs in place for splicing
Describe the steps that occur in the maturation of an RNA to form an mRNA and
allow it to be translated.
Pre-mRNA –> 5’ capping, 3’ polyadenylation, Splicing –> translatable mRNA
must go through some processing.
- 5’ Capping: cap 5’ end with an atypical nucleotide (7-methylguanosine) by 5’-5’ triphosphate bridge. Occurs long before RNA Pol has finished transcribing full gene.
- 3’ Polyadenylation: provides newly transcribed mRNA with a long 300bp polyA tail (Adenine nucs)
- Splicing: introns are removed as lariats from the coding sequence (exons) by snRNPs–complex of small nuclear RNA and protein via 2 transesterification rxns
Outline why these steps of RNA processing occur
Why process the primary RNA Transcript?
- stability of mRNA
- export from nucleus to cytoplasm
- to mark it as completed
- translational signal
- prevents the cell from translating partial fragments of mRNA
- protection from exonucleases (degradation)
- Splicing increases the coding capacity of the genome
What is a utility of introns?
They provide the opportunity for differential splicing –aka, the mRNA can be assembled in different ways generating functionally related but distinct gene products
especially for creating tissue-specific forms of a protein
Describe alternative splicing and outline models for how it might occur.
Self-Splicing Introns
- an example of RNA having catalytic potential: pre mRNA in protozoa, mitochondria, chloroplasts catalyze their own conversion to RNA
- another: ribozymes – catalyze the cleavage of other RNA molecules (RNA polymerization, alkylation, DNA ligations, etc)
Describe how problems in splicing can result in disease.
Hemoglobin deficiency caused by abnormal splicing of Betaglobulin RNA, a subunit of hemo
Problem 1: Defect in 3’ Splice Junction at end of 2nd intron
Problem 2: Mutation at 3’ Splice Junction at end of 1st intron
3 main sequences in splicing
5’ splice junction
Branch point – absolutely essential
3’ splice junction
Describe the properties of the genetic code and provide a rationale for each of
these properties.
- Universal –all derived from one common ancestor; found in all organisms
- Non-overlapping – if it did overlap, there would be restrictions on which codon could come next
- No gaps –no introns, no blank space
- Redundancy – some codons specify the same aa (usually happens in 3rd position = WOBBLE POSITION). (61 codons for 20 amino acids) (3 stop codons, 1 start codon)
- But amino acids found less frequently in proteins have less codons
- Functionally related aas have similar codons(because increases chance of single base mutation still being a functional protein)
Describe the structure/function relationships of tRNAs.
Structure: - ~80 nucs in length, cloverleaf sec structure (called stem-loop, looks like L) bc of base pairing to itself causing double helical regions. Have some unusual bases caused by chemical modifications after its been synthesized.
Function: Molecular adaptors that link amino acids to codons (bring aas to growing peptide chain).
Each tRNA has its own anticodon, and thus matching aa.
Their anticodon region hybridizes with the codon, and the right aa is covalently linked to its 3’ end
Which mutation is least deleterious to the function of a protein?
Insertion of 3 consecutive bases to a gene. because it doesn’t change the frame, just adds an aa.
Define the different mutation types:
Missense
Nonsense
Frameshift
Missense: single base CHANGE –> one diff aa
Nonsense: ^^ but results in a stop codon
Frameshift: insertion or deletion of a base –> changes the whole sequence of aas from there on
