Final Exam Flashcards
How is translation terminated in bacteria?
If stop codon occupies A site:
- RF1/RF2 bind to stop codon, cause polypeptide termination
- RF3-GDP binds to RF1/RF2, hydrolyzes to GTP causes release of RF1/R2
- RF3-GTP hydrolyzes to GDP causes release of RF3
How is a ribosome recycled?
- Release of polypeptide chain occurs with uncharged tRNAs occupying P&E sites
- Ribosome recycling factor (RRF) binds A site
- Ribosome-bound RRF recruits EF-G-GTP to stimulate release of uncharged tRNAs in P site
- Hydrolysis of GTP gives the energy to translocate ribosome and eject one of the uncharged tRNAs
- EF-G stimulates translocation of RRF to P site triggers release of RRF and EF-G
- Now empty ribosome allows IF3 to bind, which makes large and small ribosomal subunits to separate and release mRNA
How does puromycin act as an inhibitor of translation?
Puromycin acts as a new AA, tricks ribosome to stop synthesis right where it binds causing termination of translation, but it doesn’t attract RF so ribosome can’t break down to make more proteins
If a ribosome lacks stop codon it cannot recruit RF. How does tmRNA rescue stalled bacterial ribosomes?
tmRNA resembles tRNAala with extra codons and a stop codon.
- Translocation moves original mRNA out and tmRNA to P site
- Translation resumes through 9 more codons encoded by tmRNA, then terminates at new stop codon.
- The 10 amino acids encoded by tmRNA are recognized by cellular C-terminal specific proteases
If a ribosome lacks stop codon it cannot recruit RF. How does non-stop mRNA decay rescue stalled bacterial ribosomes?
- Eukaryotes have a polyA tail so, won’t hit 3’ end until translating a ton of Lys.
- Ski7 recognizes lascent synthesis of poly-Lys
- Ski7 triggers dissociation of ribosome by recruiting exosome
- Cellular protease that recognizes poly-Lys degrades mutant protein
What do EJCs do?
- Splicing machinery leaves protein complex at each exon junction
- First read of mRNA by ribosome displaces EJCs
- In correct transcription, the stop codon will come after the final EJC
- If EJC is downstream from stop codon that means the extra protein was supposed to be translated
An extra EJC is a marker of early stop codon, how is this fixed?
- Early stop recruits UPF1 & UPF2 which chew up defective protein
- UPF1 & UPF2 recruit decapping enzyme which removes the 5’ cap to prevent eIF4f from loading. 5’ cap removal exposes mRNA to exonuclease
How are polypeptides translocated into the ER in eukaryotes?
- Some proteins are transported across the ER membrane during translation. These proteins have a signal sequence that binds to signal recognition particle (SRP)
- First 20 amino acids are postal code
- SRP looks for specific postal codes than looks for its receptor
- SRP binds SRP receptor which binds translocon (polypeptide channel)
Why are all genes not expressed in every cell at all times?
DNA to protein takes a lot of time so there is a balance between wasting energy, but also being able to respond quickly
What are transcription factors and what do they do?
Activators and repressors bind to a sequence of DNA to change the affinity of the RNA polymerase for a promoter
What are enhancers? “DNA in the football feild can decide if we make RNA at the chair”
- Regulatory sites for eukaryotic genes that are 1000’s of bp away from core promoter
- Sequence orientation does not matter
- Enhancers can bind activators or repressors
What do architectural regulators do?
Bend the DNA complex to promote looping –> brings the enhancer close to the promoters
What are coactivators and corepressors?
Coactivators and corepressors bind to TF to act as a bridge between things. Whatever is on top wins
What do insulators do?
Insulator prevents cross-talk among regulators or promoters: keeps an activator or repressor from talking to its neighbor
What is signal integration? What is an example?
Control of a gene by multiple regulators in response to more than 1 signal.
Ex: Glucose is the preferred sugar, so if both glucose & lactose are available, the lac promoter is not activated
What is an effector?
Small molecule or another protein that binds an activator or repressor and causes a conformational change that increases or decreases its function
What is a regulon? What do they do?
Many operons with common regulator. Regulons allow for shifts in cellular function that can require the action of hundreds of genes: ex- multiple genes turned on by the presence of same activator
What is an operon?
genetic regulatory system found in bacteria in which genes coding for functionally related proteins are clustered along the DNA.
What is combinational control in gene regulation? (Lock & Key model)
Using multiple TF for every gene in a genome would be energetically costly so we use different combinations of a limited set of TF to differentially regulate many genes.
What does it mean that eukaryotic transcription is repressive?
Nothing happens if nothing is bound
How can a TF recognize DNA?
Limited set of known structural motifs have a recognition helix that read out bases through major grooves.
What are dimers?
Activators and repressors are usually dimers: the 2 subunits bind cooperatively at an inverted nucleotide sequences
What are motifs?
A part of protein with distinct structure that is functionally separable from the rest of the protein to read out DNA
What is the helix-turn-helix DNA-binding motif?
- 2 short a helices connected by B turn
- only red helix reads out DNA
- by itself does not function as TF
What is the basic leucine zipper motif?
bZIPs grip DNA as tongs
What is the zinc finger motif?
-Zn2+ ion does not directly interact with DNA; it stabilizes motif to allow it to present recognition helix to DNA
What is linker scanning?
Used to determine which DNA sequences will recruit a TF. The more mRNA, we can assume it recruited more activation TFs.
What was the experiment demonstrating the separate DNA-binding regulatory domains?
Combine eukaryotic activator with a bacterial repressor and make a fusion protein and you should see activity
How many motifs do TF have?
2 motifs - one to bind DNA and one to recruit polymerase
What is RNAseq?
Identity and state of cell can be read out through chopped up mRNAs and put into sequencer. New way: isolate cells and put a unique barcode on every sequencing read
Why is regulating transcription initiation most common in bacteria and eukaryotes?
Eliminates wasteful investment of cellular energy in mRNA and protein synthesis. Regulation downstream can respond more rapidly
How is translation initiation regulated in eukaryotes?
Repressors that disrupt eIF4G bridging the 5’ cap and 3’ polyA tail.
What are the 3 main ways cells send signals?
- Dimerization
- Phosphorylation
- Translocation
What is the long term response on insulin?
- Insulin receptor is a dimer that spans from outside the membrane to the cytoplasm
- Insulin stimulation increases glucose uptake by the cell
- IRS-1 insulin stimulation receptor substrate
- No change in mRNA or protein synthesis needed
- Activates gene expression to express more receptors
What is the short term response of insulin?
End result: Taking glucose transporter and transporting to cell membrane
What is the protein ubiquitnation pathway?
- E1, E2, E3 cascade links target protein to ubiquitin
What is the N-rule?
Adding ubiquitins on to the N-terminal. -can be added in chains: polyubiquitin
What is the structure of the proteasome?
Multi-subunit barrel is the core - each end of core capped by regulatory particle with 4 functions:
- bind polyubiquitin linkage on target protein
- unfold target protein
- transfer unfolded protein to central chamber “death chamber” of core
- remove ubiquitin from target protein as it is transferred
What is the lac operon?
Encodes lac repressor and lac repressor binding site
How is lactose metabolized in E.coli?
- need cell machine to cut lactose in half to eat
- presence of allolactose renders the repressor non-functional
What was Jacob and Monod’s experiment of the lac operon?
Experiment: heterozygous expression of wild-type and mutant lacI loci
Result: Both sets of lactose metabolism genes are repressed normally
Conclusion: LacI produces a diffusible product that can act as DNA in trans, while lacO controls gene expression in cis
What happens in the absence of lactose?
Lac repressor binds lacO to block initiation by RNA polymerase
What is the interaction of the Lac repressor and the operator region?
4 total LacI proteins are needed to repress on the operators.
2 TF on O1
2 TF on O2/O3
What is IPTG?
IPTG is an inducer of the lac operon to control timing of transcription at our discretion
What is cAMP and CRP?
cAMP: small molecule effector of lac –> activator= cAMP receptor protein (CRP)
What is the role of cAMP and CRP in activating the lac operon?
CRP binds to DNA with high affinity when it is bound to cAMP, low affinity when unbound.
*cAMP levels are controlled by glucose levels
Glucose high, lactose low:
Glucose low, lactose low:
Glucose high, lactose high:
Glucose low, lactose high:
How is the ara operon regulated?
- AraC is a repressor of the ara-operon, repressing transcription when arabinose is absent, AraC dimer loops DNA to block RNAP
- Convert AraC from repressor to activator depending on prescence of arabinose (=effector)
- Activation of ara operon also requires cAMP-CRP
How is the gal operon regulated?
- Gal repressor only binds when galactose is absent and causes RNAP to fall off
- Gal sugar is the effector
- CRP acts as activator
high [glucose]
low [cAMP]
CRP = ?
CRP inactive
How is the trp operon regulated?
- Trp repressor binds operator when bound to its effector
- Trp repressor binds when Trp is high because there is no need for additional synthesis
- “leader sequence” at beginning of transcript controls how much is made
What do the 4 functions of the leader sequence do?
- When trp is high: Regions 3&4 bp to form a terminator causes polymerase to fall off before
- When trp is low: Regions 2&3 can bp to make genes downstream.
- Affinity for 2&3 bp is higher
What is the SOS response?
normally lexA acts as a repressor and prevents transcription of SOS genes, but when DNA damage occurs RecA protein binds and causes auto cleavage of LexA
What happens when there is a low abundance of amino acids resulting in a high abundance of tRNAs?
Stalling ribosome binds to RelA when uncharged tRNA occupies Asite which catalyzes synthesis of ppGpp to inhibit transcription of rRNA genes.
What is a riboswitch?
A piece of RNA on the mRNA that has secondary structure that can bind an effector, causing a change in mRNA transcription or translation
What is the ligand specificity in the glmS riboswitch?
glmS pre-mRNA has ribozyme activity: it cleaves itself when bound to GlgN6P and cannot substitute ligands
What is heterochromatin? Euchromatin?
Heterochromatin: Nucleosomes are tightly packed together, no access to RNAP
Euchromatin: Nucleosomes are spaced apart; have access to RNAP
What are post-translational histone modifications?
Regulation of switching between the 2 chromatin states by change charge of histones (adding charge = spaced, removing charge = condense)
What is HAT?
Histone acetyl transferase- adds acetyl groups to histones to cause chromatin opening
What does the SWI/SNF complex do?
Remodels chromatin by binding and forcefully pushing/pulling histones around to bury or expose promoters
What is the pairing relationship between the amino acid and tRNA?
Each amino acid has a specific tRNA aminoacyl synthetase that regocnizes all tRNAs that base pair with codons for that amino acid
What are the wobble base pairings?
- G to U
- A to G
What are the functions of the different subunits of the ribosome?
Small = decoding Large = Peptide bonds
What is the Shine-Dalgarno sequence? What is the Kozak consensus sequence?
Shine Dalgarno: Nucleotides upstream of 5’ AUG to distinguish where to start ORF in bacteria
Kozak: equivalent in eukaryotes
What is the fMet-tRNA^fMet (bacteria)? tRNAi^Met (eukaryotes)?
Exclusively used to recognize the start codon and only bp with 5’ AUG downstream of Shine Dalgarno sequence
What is the process of translation initiation in bacteria?
- IF1 blocks tRNAs from binding A site. & IF3 blocks premature combination of subunits
- fMet-tRNAfMet accompanied by IF2-GTP bp with the start codon in P site
- The large subunit associates as IFs leave due to hydrolyzing GTP
How is translation initiated in eukaryotes?
- tRNAiMet is brought in before mRNA
- eIF4F grabs onto mRNA and loads it into small subunit
- Large subunit associates
- eIF4F stays bound to mRNA again to bridge 5’ cap and polyA tail
What are the steps of translation elongation?
- EF-Tu - GTP brings new amino acid tRNA to A site
2. EF-Tu releases upon hydrolyzing GTP
What is a mediator?
Multi-protein complex that functions as an activator at most promoters
What do Gal4p, Gal80p, and Gal3p do?
Gal80p (corepressor) binds Gal4p (activator) to prevent its activation of Pol II in the absence of galactose. Gal3p + galactose present switches Gal80p from a corepressor to co activator.
What is the role of galactose in regulation of Gal genes?
Galactose is a small effector for Gal3p causing it to bind to Gal80p
What is the role of Mig1?
Mig1 is recruited when there is a lot of glucose to repress Gal4p
What is combinatorial control by heterodimer?
Bacterial TF come as homodimers, but some types of eukaryotic TFs can function as heterodimers. Different heterodimer combinations have different DNA-binding affinities and different activation potential
