1
Q
how are Both prokaryotes & eukaryotes affected by antibiotics and toxins?
A
Ribosomes and their components are frequent targets for antibiotics:
-Antibiotics can block exit channels of ribosome
->Can interfere with normal function of accessory factors
(EF-Tu, EF-G)
->Common antibiotics streptomycin, neomycin, gentamycin, tetracyline, spectinomycin interact with small ribosomal subunit, erythromycin & others with large subunit to induce mistranslation or block charged tRNA binding
-Toxins can chemically modify ribosome components
2
Q
role of Puromycin in Interfering With Translation?
A
antibiotic that terminates translation by mimicking a tRNA in the A site
3
Q
How can you control the amount of a protein in the cell?
A
1) Usual way is to alter transcriptional rate.
2) Another way is to alter the translational rate/protein synthesis, bypassing need to…
a) transcribe RNA (ATP, GTP!)
b) process mRNA (more ATP)
c) shuttle mRNA to cytoplasm from nucleus (more GTP)
* can respond rapidly to external stimuli->Cells can use pre-made mRNA to rapidly make protein on demand
4
Q
describe how the primary target of regulators (of bacterial translation) is to interfere with the recognition of the RBS by the 30s subunit (2 ways)
A
- RNA-binding proteins will bind next to RBS and thus prevent 30S binding of 16s rRNA
- >don’t target mRNA’s RBS directly due to RBS site conservation (this stops 30S subunit from binding all mRNAs!) - RNA molecules can themselves block translation by self-binding (mRNA base pairs with itself to mask RBS), usually in polycistronic mRNA -> prevents later ORF translation until earlier ORFs are translated and “unmask” later ORFs (mRNA no longer self bound)
5
Q
how can Bacterial ribosome synthesis be regulated at level of translation
A
Ribosomal Proteins Are Translational Repressors of Their Own Synthesis
-Autorepression of ribosomal protein occurs -> repressors bind their own mRNA near RBS, prevent translation
6
Q
explain how ribosomal protein synthesis is inhibited only when the regulatory/repressor ribosomal protein is in excess
A
Available ribosomal proteins find and bind the corresponding available rRNA at high-affinity sites which allows proper folding of rRNA & proteins into ribosome complex (allow proper assembly of ribosome)
-if all the rRNA binding sites are occupied, then ribosomal protein will find and bind a “second choice”binding site on their own mRNA (has lower affinity for this mRNA than for rRNA) and this prevents the translation of the ribosomal protein by inhibiting translation of the open reading frame
7
Q
how do Eukaryotes block the two initiating events of translation if under stress?
A
under conditions of reduced nutrients or other cell stresses, it is often useful for Eu cells to reduce translation globally. in these instances, two early steps in Eu translation initiation are targeted for inhibition:
- recognition of the mRNA
- targets the 5’ cap-bindng protein eIF4E which later binds to eIF4G. eIF4E-bindinf proteins compete with eIF4G for binding to eIF4E. In unphosphorylated state, eIF4E-BP bind to eIF4E tightly and inhibit translation. 4E-BP phosphorylation is mediated by protein kinases called mTor. (inhibitors of mTor are affective chemotherapy agents) - Initiator tRNA binding to the 40S subunit
- inhibition mediated by phosphorylation of eIF2. eIF2 bound to GTP is required to deliver initiator tRNA to P site of ribosome. phosphorylation causes reduced levels of eIF4-GTP which is needed to transport tRNA and therefore translation initiation inhibited
8
Q
ferritin is an Fe2+-binding protein that stores iron and controls iron release and is the major regulator of iron levels in the human body. therefore ferritin must be quickly made to respond to iron levels and so transcriptional control is required. How is Ferritin translation regulated ?
A
Ferritin transcription is regulated by iron-binding proteins called iron regulatory proteins (IRPs). these proteins are also RNA-binding proteins that recognize a specific hairpin structure formed in ferritin mRNA called iron regulatory element (IRE)
- In cells with little iron, the conc. of iron too low to bind IRPs. with no iron bound to the IRPs, they instead bind to the IRE and inhibit the ability of eIF4A/B to unwind hairpin structure which prevents translation from occurring.
- > when conc. of free iron in the cell is elevated, the IRPs bind iron and the IRPs lose ability to bind to the IRE and therefore can’t inhibit translation.
9
Q
explain how Translation of Yeast Transcriptional Activator Gcn4 Is Controlled by Short Upstream ORFs and Ternary
Complex Abundance in starvation and non-starvation conditions
A
Gcn4 controls transcription of many amino acid biosynthesis enzymes (Low amino acids -> more Gcn4 -> more A.A. biosynthesis enzymes -> more A.A.s! OR High A.A. -> less Gcn4 -> less enzymes…)
Nonstarvation conditions: Gcn4 not translated
- Gcn4 mRNA has 4 small ORFs called uORF 1-4 that are upstream coding sequence for Gcn4
- once 40S ribosome recognizes first uORF (uORF1), ribo remains bound to RNA and continues scanning for downstream AUG codons.
- The ternary complex (eIF2-GTP + initiator tRNA) needed for scanning, rebinds to the ribo after uORF1 is translated. Once one of the other (inhibitory) uORFs are translated, the ribo dissociates and falls off and Gcn4 is not translated.
Starvation conditions: Gcn4 is translated
-Low A.A. levels means a lot of uncharged tRNAs with
no A.A.s to carry
-activated eIF2 kinase (Gcn2) phosphorylates eIF2 and slows rate at which eIF2 can bind GTP
->Less eIF2-GTP means that the 40S subunit will slows down ternary complex binding due to rarity, can’t bind mRNA without it -> just continues scanning along and misses start codons after uORF1…
->uORFs #2-4 never made, but Gcn4 start codon is recognized and Gcn4 translation occurs (bc it is likely that the Ternary complex will rebind ribo before it reaches Gcn4 encoded region)
10
Q
how do Prokaryotes rescue ribosomes on defective and thus dangerous mRNAs?
A
Prokaryotes rescue ribosomes with by terminating defective mRNAs with special tmRNA: part tRNA, part mRNA
tmRNA = transfer-messenger RNA
11
Q
what is SSrA?
A
a 457-nucleotide tmRNA that includes a region at it’s 3’ end that strongly resembles tRNA^Ala
- > SSrA can therefore be charged with alanine and bind EF-Tu-GTP. this complex binds to A site of ribo (tmRNA can only do this due to empty A site-> only then is there room for tmRNA tail) and participates in peptidyl transferase reaction and peptidyl-SSrA translocation results in the release of the broken mRNA and then the SSrA acts like mRNA by entering mRNA binding channel of ribo and encodes 10 codons followed by a stop codon.
- > the protein coded by the incomplete mRNA is fused to the 10-amino acid peptide tag which causes protein to be rapidly degraded (prevent from damaging the cell)
12
Q
what are the two options eukaryote cells have to degrade mRNAs that are incomplete or have premature stop codon
A
1) nonsense-mediated mRNA decay (premature stop codon-> frameshift error)
2) “non-stop”-mediated mRNA decay (no stop codon)
13
Q
describe Nonsense mediated mRNA decay
A
- uses recruited decapping enzymes
- Relies on exon-marking proteins that are usually displaced by ribosome
1. Exon marking complexes recruit Upf$ proteins
2. Upfs interact with ribosome and activate a decapping enzyme
3. 5′ cap on mRNA is rapidly removed
4. mRNA is degraded by a 5′ -> 3′ exonuclease that attacks uncapped mRNA
14
Q
describe Non-stop mediated mRNA decay
A
- removes mRNA and protein from incomplete transcripts
- Ribosome translates polyA tail since there is no stop codon (abnormal!)
- This adds multiple lysines to protein (K=AAA, poly-Lys proteins are unstable, rapidly destroyed by proteases)
- Stalled ribosome is rescued by special eukaryotic release factor 3 (eRF3)-like proteins (Hbs1, Dom34, Ski7 but know them as “eRF3 like H-D-S proteins”)
- Ribosome is released and exonucleases are recruited
- mRNA is degraded by exonucleases
15
Q
Codons specifying the same amino acid are called ?
A
synonyms
16
Q
how does the code seems to minimize effect of any mutation?
A
- Mutating first position of a codon often gives a similar, if not the same amino acid
- Codons with pyrimidines in 2nd position specify mostly hydrophobic amino acids
- Codons with purines in 2nd position correspond mostly to polar amino acids.
- therefore transitions usually replace one aa with a very similar one.
- Change in the 3rd position rarely results in a different amino acid, even due to transversion
17
Q
explain Wobble in the anticodon
A
- base at 5′ end of anticodon is not as spatially confined as the other two..- > allows 5′ end of anticodon to form hydrogen bonds with more than one base located at the 3′ end of a codon
- Early work found tRNAs could bind more than one codon because fifth “base” was found in tRNA anticodons, Inosine (made from deaminated adenine, really a nucleoside)
- Inosine (I) in anticodons can pair with several different bases in codon (can wobble between them)
- Non-canonical Watson-Crick base pairing also occurs (G-U)
- wobble explains why tRNAs don’t exist 1 per codon
18
Q
Why is the Wobble allowed at the 5′ Anticodon?
A
3-D structure of tRNA shows stacking interactions between the flat surfaces of 3 anticodon bases and 2 following bases
- These position the first (5′) anticodon base at the end of the stack, thus less restricted in its movements
- The 3′ base appears in the middle of anticodon loop stack, resulting in the restriction of its movements
- The 5’ anticodon wobble base moves free since it is at the end of base pair stack and therefore allows tRNA to recognize a maximum of 3 codons (only when Inosine is at 1st (or 5′) anticodon position)
- adjacent base to the third (3’) anticodon base is always a bulky modified purine residue which further restricts ots movements and explains why wobble is not seen in the first (5’) position of the codon
19
Q
Three Rules Govern the Genetic Code?
A
- Codons are read only in a 5′ to 3′ direction (to properly define peptide sequence)
- Codons are nonoverlapping and the message contains no gaps.
- The message is translated in a fixed reading frame, which is set by the initiation codon.
20
Q
What are the 3 kinds of Point Mutations that alter the Genetic Code?
A
missence, nonsenese and frameshift mutations
21
Q
describe:
Missense mutation, Nonsense or stop mutation and a Frameshift mutation
A
Missense mutation
= an alteration that changes a codon specific for one amino acid to a codon specific for another amino acid (can be silent)
E.g., Sickle cell anemia caused by hemoglobin E -> V mutation
Nonsense or stop mutation
=An alteration causing a peptide chain-termination codon.
Frameshift mutation
= insertions or deletions of one or a small number of base pairs that alter the reading frame
22
Q
Suppressor mutations?
A
Suppressor mutations (partly) reverse a mutation by another elsewhere
- > They suppress the change due to mutation at site A by producing an additional new genetic change at site B
- > Suppressor Mutations Can Reside in the Same or a Different Gene (site B is can be in the same or a different gene)
23
Q
Intragenic suppression vs Intergenic
suppression mutation?
A
intragenic= occurring within the same gene as original mutation, but at a different site
intergenic = occurring in another gene
24
Q
Reverse (back) mutations?
A
change an altered nucleotide sequence back to original sequence
e.g., reverse the harmful mutations by a second genetic change
25
Intragenic suppression examples?
1) missense mutation - original mutation at site A can be reversed through 1 more missense mutation in same gene
2) frameshift mutation – add/delete base to put ORF back into frame again (Protein reverts almost back to normal)
26
Intergenic suppression mutation example?
Nonsense Suppression
- Mutant tRNA genes suppress effects of nonsense mutations in protein-coding genes-> Mutated tRNAs can rescue nonsense mutations by introducing an amino acid instead of terminating the chain
- They act by reading a stop codon as if it were a signal for a specific amino acid.
- The act of nonsense suppression is a competition between the suppressor tRNA and the release factor
27
Genomics revolution has confirmed the universality of the genetic code..…with the exception of ??
mitochondrial DNA, the genetic code is slightly different from the standard code
->mitochondrial tRNAs are unusual in how they decode mitochondrial mRNA
-Only 22 tRNAs found in mammalian mitochondria
because the U in the 5′ wobble position of a tRNA is capable of recognizing all four bases in the 3′ of the codon
(instead of just 3 in universal code…)
->Some bacteria, protozoa, and eukaryotes have non-standard codes too
28
Gene expression is controlled by regulatory proteins. whats the difference between positive and negative regulators?
- positive regulators, or activators increase transcriptional rate, negative regulators, or repressors decrease transcriptional rate
- both are DNA binding proteins that recognize specific sites at or near the genes they control
- which steps in transcription that are stimulated by activators and inhibited by repressors depends on the promotor and regulators in question
29
why do most activators and repressors act at the level of transcription initiation? what is a reason for regulating at a later step?
it is the most energy efficient step tp regulate (makes sure no energy in wasted in making mRNA that doesn't end up getting use/translated)
-also, regulation at first step is easier to do (only a single copy of each gene for haploid genome, so only a single promotor on a single DNA molecule needs to be regulated to control expression of a given gene)
However, also good to regulate at later steps too because it allows more inputs (more signals can modify its expression if a gene is regulated at more than 1 step), it also decreases response time (dont have to transcribe/process mRNA before protein is made)
30
what is the basal level of transcription ?
RNA Pol binding to “naked” promoter is usually weak in the absence of regulator proteins
- > spontaneously change to open complex
- > start low-level transcription
31
how are promotor sequences on DNA regulated by regulatory proteins?
Repressors bind and block “operator sites” (prevent RNA Pol from binding to the DNA)
-Some activators bind DNA site and then help RNA Pol bind promoters, i.e., recruitment (an examples of cooperative protein binding to DNA)
32
how do some activators work by allostery and regulate steps after RNA polymerase binding?
Some promoters require activators to stimulate the transition from closed to open complex
->Activators stimulating this type of promoter function by triggering a conformation change in RNA Pol or DNA itself
33
Activators can change DNA or RNA Pol conformation to “open” by allostery, give an example of both
1. The NtrC (Nitrogen regulatory protein C) activator binds with RNA Pol protein, stimulating a change from a closed
to an open promoter complex
2. MerR activator binds to merT gene promoter to cause DNA to change conformation -> leading to activation
34
Cooperative binding between gene regulatory proteins at adjacent sites is common but how come regulatory proteins don’t need to be immediately next to each other for function?
distant DNA sites can be brought closer together to help looping with a DNA-bending protein (i.e., “architectural” proteins)
ex. NtrC activates a promoter “from a distance” since its binding sites are ~150 bp upstream of the target promoter (can be artificially placed 1kb away)
35
Simple vs complex cooperative binding of regulatory proteins
Simple cooperative binding: activator interacts simultaneously with DNA and RNA polymerase to recruit RNA Pol to the promoter
Complex cooperative binding: Can also have activator (or more than one!) bind and activate RNA Pol that is already bound to promoter
36
Allostery not only a gene activation mechanism, it is also a way regulators are controlled by their specific signals, how?
A typical bacterial regulator can adopt 2 conformations- one can bind DNA, the other cannot -> dependent on signal molecule binding to it which locks the regulatory protein in one or the other confirmation, therefore determining whether or not it can act.
37
briefly describe the classic example of Bacterial Lac operon gene regulation by repressors and activators
lac operon has 3 (structural) protein-encoding genes for lactose metabolism:
1. beta-galactosidase (lacZ) that breaks down lactose
2. galactoside permease (lacY) for transporter to uptake lactose
3. galactoside transacetylase (lacA) to destroy toxic lactose analogs
* 3 lac genes transcribed together on 1 polycistronic mRNA from lac promoter
Lac operon controlled by type of sugar present in cell through two regulatory proteins:
1. Catabolite Activator Protein (CAP) A.K.A. CRP (cAMP receptor protein)
- > binds cAMP, then lac promoter to promote transcription (low glucose causes ATP depletion, so cAMP builds up and binds CAP, then CAP-cAMP binds promoter)
2. a repressor protein called the Lac repressor which is encoded by the LacI gene located near the other lac genes (lots of lactose -> binds to LacI, “de-represses” gene, LacI-lactose releases promoter)
38
what is the Mechanism of Repression by the lac repressor? why does RNA Pol needs CAP to bind promotor efficiently?
Lac operator is made of “half-sites” for RNA Pol binding
(“Half-sites” are mirror images of other)
-The lac operator overlaps promoter so any repressor bound to the operator physically blocks RNA polymerase from binding
-RNA Pol needs CAP to bind, interacts with it since no UP element here
-CAP site is similar to lac operator site-> almost a direct (inverted) repeat
39
what is the activating region of CAP ?
CAP has an activating region but it isn’t part that binds DNA but interacts with the α-subunit of RNA polymerase
-Carboxyl-Terminal Domain (CTD) of RNA Pol is a CAP-binding domain
40
how do Lac regulators invade and bind DNA ?
by forming specific H-bonds with it:
CAP and lac repressor bind DNA using a common structural motif.
-Recognition of specific DNA sequences is achieved using a conserved region of a helix-turn-helix
-This domain is composed of two alpha helices, one is the recognition helix -> Recognition domain slips into major groove and binds to DNA bases
-Specific amino acids interact with specific hydrogen bonds of DNA bases
Lac repressor is similar but binds as tetramer (not a dimer) to main lac operator and also another minor one
41
how are the activities of Lac repressor and CAP controlled allosterically by their signals?
- It is allolactose that controls Lac repressor (not lactose itself) by binding to Lac repressor LacI-> triggers change in the LacI protein’s conformation (shape) and LacI falls off DNA
- Glucose lowers the intracellular concentration of a small molecule, cAMP (more ATP, less cAMP), cAMP is the allosteric effector for CAP-> Only when CAP is complexed with cAMP does CAP-cAMP adopt a conformation that binds DNA
* Some transcripts do sneak even with the presence of the lac repressor-> ensures a very low level of Lac proteins (otherwise, how would lactose get into cell if not for permease?)
42
why do alternative sigma factors direct RNA Pol to alternative promoters? 3 examples
1. under certain conditions sigma factors direct RNA Pol to specific promoters other than the lac promotor
-Heat shock sigma 32 factor translated better when E. coli subjected to heat shock, the amount of this new sigma32 factor increases in the cell, and displaces sigma70 from a proportion of RNA polymerases
2. -Sigma54 is required to transcribe Ntr genes involved in nitrogen metabolism
3. -Viral phage SPO1 gene transcription in infected B. subtilis cells proceeds according to a temporal program where sequential gene transcription occurs with early genes first, then middle genes, and finally late genes
Switching is directed by phage-encoded σ factors associated with host core RNA Pol
*Sigma Factor swapping allows regulation of which bacterial genes are transcribed (directs enzymes to transcribe genes whose products will protect the cell from the effects of heat shock, etc.)
43
The majority of activators work by recruitment - 2 exceptions?
NtrC and MerR: transcriptional activators that work by allostery rather than by recruitment
-RNA Pol first binds the promoter in an inactive complex, then activator triggers an allosteric change in that complex
-Nitrogen metabolism NtrC induces a conformational change in RNA Pol to form an open complex
(controls glnA gene which has prebound RNA Pol)
-mercury-resistance MerR protein controls merT enzyme transcription, MerR protein will be prebound to DNA and waits for RNA Pol to be bound, and opened (allosteric effect of activator on DNA instead of RNA Pol)
44
how does the transcriptional activator NtrC work? where/how does it bind?
NtrC is modified to bind DNA before using ATP to activate RNA Pol:
NtrC activated and bound to site when low nitrogen stimulus occurs, becomes phosphorylated by NtrB kinase which reveals its DNA binding domain
-NtrC binds each site as a dimer, and interacts with other dimers, binds the 4 sites in a cooperative manner
-NtrC then interacts directly with σ54 causing a RNA Pol
conformational change using ATP energy (DNA has to loop to bind σ54)
*Bending protein may be needed for some NtrC-controlled genes
45
how does the transcriptional activator MerR work?
MerR conformational change shortens & rotates promoter to activate gene:
- MerR binds to merT promoter DNA between the -10 and -35 regions (binds to opposite face of the DNA helix from that bound by RNA Pol )
- MerR binds Hg2+ and undergoes conformational change that causes the DNA in the center of the promoter to twist. this shortens distance between -35 and -10 sequences to allow merT transcription
* MerR doesn’t interact directly with RNA Pol!
46
E. coli araBAD operon promoter is activated by arabinose sugar and the absence of glucose and directs expression of genes encoding arabinose metabolism enzymes. How is it regulated by AraC?
Activator AraC adopts different conformations in the presence or absence of arabinose (works with CAP)
- When AraC binds arabinose, AraC dimerizes to bind araI “half-sites”
- With no arabinose to bind, the AraC protein changes shape and binds only 1 araI half site, and 1 distant araO(perator)2 site (araBAD genes not expressed)
- Large activation of transcription by adding arabinose-> used in plasmids
* *AraC binding of arabinose yields an activator, arabinose loss gives antiactivator
47
describe the two ways Bacterial virus Phage/bacteriophage lambda can replicate
can replicate in either of two ways: lysogenic and lytic (lysis)
- Lysogenic growth = phage DNA passively replicating as part of host DNA
- Lytic growth= requires replication of the phage DNA and synthesis of new coat proteins
- Bacterial viral propagation can proceed silently or destructively
48
lysogens?
bacteria with integrated phage DNA
49
prophage?
Integrated viral DNA into host genome
50
lysogenic induction?
prophage switches from lysogenic to lytic growth
| induced by DNA damaging agents that threaten the host cells continued existence…
51
describe the three promotors in the control region of λ DNA that control type of λ propagation (whether virus DNA replicates lysogenically or lytically)
λ DNA has genes for DNA replication & recombination and coat proteins and the promotors in the control region dictate what proteins are made
-PR (rightward promotor) and PL (leftward promotor) are strong promoters, do not need activator
-PRM (Promoter for Repressor Maintenance) transcribes only cI lambda repressor gene-> only directs efficient transcription when a needed activator is bound just upstream (weak promotor)
*PL and PR-driven protein synthesis leads to cell lysis by making lytic proteins (lytic growth)
*when PL and PR are shut off, there is no lysis since
PRM drives synthesis of cI repressor protein (lysogenic growth)
*Switch from one promoter set to another dictates λ growth proteins made
52
cI gene encodes λ phage repressor, what is it?
= a protein of 2 domains joined by a flexible linker region
- also an activator (despite the protein’s name!)
- As an activator, lambda repressor works like CAP, by recruitment of RNA Pol
- λ repressor’s activating region recruits RNA polymerase’s σ subunit
53
what is the Cro gene of bacteriophage control region?
Cro (Control of repressor and other things) only represses transcription (no activation)
54
λ repressor & Cro can bind to any 1 of 6 operators (“left” and “right” promoters each have 3 operators)
Describe why lambda phage operators in promoters need different repressor amounts for effect
OR1,OR2, and OR3 in right operators have roughly similar sequences but strength of regulator protein binding varies between operators.
- λ repressor & Cro repressor bind each operator as dimers but need 10X’s more λ repressor concentration to bind OR2, OR3 compared to OR1 (whereas Cro binds OR3 with highest affinity)
* Cro repressor binds in opposite direction, with opposite strength
55
how does the λ repressor bind to operator sites ?
λ repressor binds to operator sites cooperatively (binding of one protein makes the binding of the next one easier)
- λ repressor at OR1 helps it bind to the lower affinity site OR2 by cooperative binding.
-> This avoids the need to have 10X’s more λ repressor protein to bind OR2 - but OR3 not helped…
-Allows any change in gene expression to be much more
sensitive to λ repressor concentration
56
how do Repressor and Cro bind in different patterns to control lytic and lysogenic growth?
During lysogeny, PRM is on, while PR and PL are off
- >The λ repressor binds to OR1 & OR2 cooperatively, but leave OR3 open
- >RNA Pol binds PRM and contacts the λ repressor bound to OR2 (activates expression of the cl(repressor) gene)
Lytic growth involves a single Cro dimer bound to OR3 (located within PRM region)
- >Cro represses the PRM promoter by overlapping it
- >This leaves RNA Pol free to bind PR and PL to drive lytic protein synthesis
57
why does Lysogenic induction require proteolytic cleavage of λ repressor?
When a lysogen’s host suffers DNA damage, the ‘SOS’ response is induced (i.e. cell infected with prophage gets sick, now time for prophage to leave this party under the threatening circumstances…)
- >The first event in SOS response is to activate RecA protein which stimulates autocleavage of LexA (repressor of DNA repair enzymes)
- >LexA repressors cut themselves in half, allowing DNA repair enzyme expression
- >Phage λ repressor mimics shape of LexA so RecA blindly activates autocleavage of λ repressor during SOS response (and seals the cell’s fate! irreversible!)
* This removes repressor from the λ operators and inducing the lytic cycle (PR, PL now free…)
58
how is λ repressor concentration tightly self-controlled for governing lysogeny?
by positive and negative autoregulation
Can’t let λ repressor level drop too low ->spontaneous lytic phase….
->That’s why λ repressor drives its own synthesis from PRM (i.e. cI gene) ->This is positive autoregulation
-If concentration of λ repressor is too high, negative autoregulation occurs by binding OR3 that displaces RNA Pol (inhibits transcription of λ repressor)
- λrepressor interacts with dimers bound to Left operators (OL1, OL2) which allows cooperative binding of λ repressor dimers to OR3 now, repressing its own transcription
-Any drop in [repressor] -> RNA Pol binds and makes more repressor!
*Negative Autoregulation of λ repressor Requires Long-Distance Interactions and a Large DNA Loop to compensate for distance
59
Another activator, λcII, controls the decision between lytic & lysogenic growth upon infection of a new host; how does it do this?
PL drives CIII stabilizer transcription ()
-CII is an activator that binds PRE (Repressor Establishment of lysogeny) that is just upstream CII and drives synthesis of cI (λ)repressor protein (
60
The number of what repressors and activators of lysogeny dictate fate of host?
CRO and CII (driven by PR)
- In early infection, CRO made and will promote lytic pathway
- Competing levels of CRO and CII dictate whether or not lytic or lysogeny is favoured
- PRE drives synthesis of cI-encoded (λ)repressor protein just like PRM does, except PRE does it to Establish λ levels
- >Eventually enough (λ)repressor protein is made to bind OR1 & OR2 sites
- >PRM then Maintains λ synthesis
61
how does the number of Phage Particles Infecting a Cell Affect Whether the Infection Proceeds Lytically or Lysogenically?
If cell infected with only 1 phage, it will likely lyse (lytic progression) vs. If cell infected with more than 1 phage, it will likely be lysogeny, therefore, the more phages that infect a cell, the more likely the cell is to survive with phage
- More phage CII activator protein is made with more phage infecting cell
- This promotes PRE synthesis of (λ)repressor protein, which will then be taken over by PRM promoter-driven (λ)repressor protein synthesis
- If one phage establishes (λ)repressor protein synthesis (and lysogeny), all other phages will too
- If many phages infect a cell, there would be a lot of competition for hosts if there was lysis -> so phage is better off waiting in cell (lysogeny!)
62
how do growth conditions of E. coli control the stability of CII protein and thus the lytic/lysogenic choice?
when the phage infects a population of bacterial cells that are healthy and growing vigorously, it tends to propagate lytically. when conditions are poor for bacterial growth, the phage is more likely to sit tight.
- If growth is good, FtsH protease is very active and degrades CII. Then CII is low, no repressor is made and cell goes to lytic pathway
- In poor growth conditions, slow degradation of CII by low FtsH activity, λ repressor can build up and leads to lysogeny
- CIII protein can also play a role -> CIII stabilizes CII protein
- CII also activates PI(ntegrase) that promotes integrase synthesis that catalyzes phage DNA integration into bacterial host’s chromosome (forms the prophage)
63
how does Transcriptional Regulation in Eukaryotes differ from prokaryotes
Eukaryotes have complex gene regulatory measures to control transcription
-Eukaryotic activators function in similar manner to
bacteri…but repressors are more complex, i.e., gene silencing
-Many eukaryotic genes have more regulatory binding sites with corresponding regulatory proteins than typical bacterial genes do (these sites may be positioned father from the start site of transcription)
-have enhancers, insulator sequences and silencers
64
what are enhancers, Insulator sequences and silencers?
- An enhancer is a unit of binding sites that binds regulators responsible for activating the gene at the given time and place (typically an enhancer regulates only one gene)
- Insulator sequences block activation of the promoter by activators bound at the enhancer (sequences found between enhancers and some promotors)
- A silencer binds regulators responsible for repressing the gene at the given time and place
65
how are Eukaryotic activators similar to prokaryotic activators in structure?
DNA-binding & DNA-activating surfaces are very often on separate domains of the protein.
66
explain the Activator Protein “Domain-Swapping” Experiments done with Gal4 and LexA
shows that activator protein domains are separable but both required for gene activation
- Gal4’s DNA-binding domain can still bind DNA by itself - but with no activation domain, it cannot activate transcription
- LexA is a bacterial repressor protein with a DNA-binding domain, binds LexA sites (LexA’s DNA-binding domain alone can’t activate gene transcription)
- Fusing Gal4’s activation domain to LexA’s DNA-binding domain confers gene transcription -> both domains required!
- lacZ is a common reporter gene (stains cells blue) that measured expression in this experiment
67
what is The (Yeast) Two Hybrid Assay (Y2H)?
- Assay used to identify proteins that interact with each other by exploiting ability to separate DNA-binding and transcriptional activation domains
- Imagine you want to prove that proteins A and B interact – even if they normally have nothing to do with transcription
- B is fused to the activation domain but can’t activate transcription by itself
- A is fused to a DNA-binding domain but can’t activate transcription by itself
- If A and B interact, they will reconstitute the DNA binding and activation domains forming a complete activator, leading to reporter gene activation
68
Eukaryotic Regulators Use a Range of DNA-Binding Domains, how does DNA Recognition compare with prokaryote regulators ?
DNA Recognition Involves Same Principles as in Bacteria:
- No great difference in how DNA-binding proteins from different organisms recognize their sites
- Eukaryotic regulators often bind as dimers, recognizing specific DNA sequences using an α-helix inserted into the major groove
- Some regulators in eukaryotes bind DNA as heterodimers (mix & match), and in some cases even as monomers
- Heterodimerization allows the cell more flexibility in controlling gene transcription
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DNA Recognition by a Homeodomain Protein
| a eukaryote DNA-binding domain
Homeodomain proteins activate genes regulating anatomy
- a class of helix-turn-helix DNA-binding domain proteins
- recognizes DNA in essentially the same way as bacterial proteins do
- > 3 α-helices where helix 3 is the recognition helix that is inserted into major groove of DNA
- discovered in fruit flies where they control many basic developmental programs
- These “homeobox” DNA site-binding proteins are found in all higher eukaryotes, also yeast (i.e., HOX genes)
- Typically bind DNA as heterodimers
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Zinc Containing DNA-binding domains
Various different forms of DNA-binding domain proteins that incorporate a zinc atom(s), i.e., Zn2+ finger (ex TFIIIA) and Zn2+ cluster
- DNA binding domains can use zinc to stabilize their structure
- ɑ-helix is the recognition helix that is againinserted into the major groove of DNA
- Recognition helix is “presented” to the DNA by the β sheet
- Zn2+ is bound by His and Cys residues found in the β-sheets that helps to stabilize the protein structure
- This arrangement stabilizes protein’s structure-> essential for binding DNA
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Leucine Zipper Motif Proteins
Leucine zipper motifs use ɑ-helix to pinch into DNA and bind each other
-This motif combines both the dimerization and
DNA-binding surfaces within a single structural unit.
-Leucine-zipper-containing proteins often form
heterodimers as well as homodimers
-Leu zipper proteins have large ɑ-helices with dimerization and DNA-binding domain at different sections along their length
->ɑ-helical coils slip into the major grooves
-two helices interact at top by hydrophobic interactions between Leu-rich areas allowing dimerization of the two dimer🤩
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Helix-Loop-Helix Proteins
Helix-loop-helix proteins exploit their loops to pack in during DNA binding
- Two helices separated by a flexible loop allowing homodimers (or heterodimers) to pack together
- Transcriptional activity often controlled at level of expression for H.L.H. protein -> 1 subunit is always present, other heterodimer subunit may only be expressed sometimes...
- Leucine zipper and HLH proteins are often called basic zipper & basic HLH proteins because α-helix region that binds DNA contains basic amino acids
- >Helical region inserts into the major groove of the DNA
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how are Activating regions defined by their makeup rather than their structure?
DNA binding domains have recognizable defined structures. In contrast, activation regions are not well-defined structures but are more variable.
->Activating regions of proteins are then grouped on the basis of amino acid content
1) Acidic activating region tend to be strong activators
2) Glutamine-rich activating region
3) Proline-rich activating region
Activation regions believed to consist of small repeating units that together are responsible for binding
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Activators help speed up transcription by bringing necessary parts together, what are the 3 ways Activators recruit RNA polymerase indirectly ?
1) Can interact with parts of the transcription machinery other than RNA polymerase itself -> by recruiting them, thus recruit RNA Pol too
2) Can recruit nucleosome modifiers that alter chromatin in vicinity of a gene and thereby help RNA polymerase bind DNA
3) Can help RNA polymerase recruit initiation and elongation factors
- >In many cases, a given activator can work in all 3 ways.
- >The eukaryotic transcriptional machinery contains numerous proteins in addition to RNA polymerase
- > contrast with bacteria!
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describe the Recruitment Of Protein Complexes To Genes By Eukaryotic Activator Proteins
Eukaryotic transcriptional machinery contains numerous proteins in addition to RNA polymerase and many come in preformed complexes such as the Mediator- and the TFIID-complex. Activators interact with one or more of these complexes and recruit them to the gene.
- High, regulated transcription levels require the Mediator Complex, transcriptional regulatory proteins & usually nucleosome-modifiers
- Mediator is associated with the CTD “tail” of the large RNA Pol subunit through one surface, while presenting other surfaces for interaction with DNA-bound activators
- Gal1 can be transcribed (activated) if LexA DNA-binding protein is fused directly to RNA Pol
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Activators also recruit nucleosome modifiers that help transcription machinery bind at the promoter on inaccessible genes packaged within chromatin. Nucleosome modifiers come in two types which are?
1. Histone acetyltransferases (HATs):
add chemical groups to histone tails to create specific binding sites on nucleosomes for proteins bearing so-called bromodomains (e.g., TFIID)
-loosen chromatin structure and frees up sites
2. ATP-dependent activity of SWI/SNF:
remodels nucleosomes to uncover previously inaccessible DNA-binding sites
*Activators reverse chromatin packing of DNA that
would otherwise hinders transcription
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how can far away enhancer regions activate promoters in eukaryotes?
- Many eukaryotic activators - particularly in higher eukaryotes- work from a distance up to 100 kb away!
- Contrast with bacteria where limit is ~ few hundred base pairs away
- Bacterial IHF architectural protein binds to DNA, bends it to help the DNA-bound activator reach RNA polymerase at the promoter
- eukaryotic DNA wrapped in nucleosomes, and histones within them are modifiable-> these affect compactness
- Therefore, DNA packing means promoter and enhancer aren’t far apart
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how/why do specific elements called insulators control the actions of activators working from distant enhancer elements ?
Insulators block activation from a distance without direct promoter binding
- >don’t want to activate nearby promoters by mistake.
- activation of the promoter by the activator bound to the enhancer is blocked by insulators despite activators binding to enhancerDNA
- The activator can activate another promoter nearby
- The original promoter can be activated by another enhancer placed downstream
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explain LCRs transcription regulation of the human globin genes
Appropriate regulation of some groups of genes requires locus control regions
- Globins are blood proteins whose expression is developmentally regulated-> fetus/child/adult -> ε (=fetus gene) - β (=adult gene)
- A group of regulatory elements collectively called the locus control region, or LCR, is found 30-50 kb upstream of globin gene cluster
- LCR binds regulatory proteins that cause the chromatin structure around globin genes to “open up”, allowing access by regulators that control expression of each genes in a defined order
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Why do activators work together synergistically to integrate signals?
Cells depend on multiple and often synergistic inputs/signals to regulate transcription and to turn a gene on. each signal is transmitted to the gene by a separate regulator, therefore at many genes, multiple activators must work together to switch a gene on.
- when multiple activators work together they often do so synergistically because the effect of two activators working together is greater than the sum of each activator working alone (usually much greater)
- Activators can recruit the same, or different parts of transcriptional machinery
- Synergy can also result from activators helping each other bind under conditions where binding of one depends on binding of the other
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how do Activators can facilitate cooperative binding through direct and indirect binding?
Direct:
1) Cooperative binding directly between two proteins A & B
2) Cooperative binding directly between 2 proteins and a third (X)
Indirect:
3) A protein (A) recruits a nucleosome remodeler that exposes a 2nd site that allows the second protein (B) to bind DNA
4) A protein (A) binds its site just near the nucleosome, causing it to unwind a little, allowing the second protein (B) to bind DNA
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Signal integration (indirect #3):
describer how HO gene transcription is dependent upon two activators working in concert
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HO gene expressed only in mother cells, only when yeast divides by budding.
HO gene controlled by 2 regulators;
1. A protein that recruits nucleosome modifiers (SWI5)
2. A protein that recruits Mediator (SBF)
* If both activators are present & active, the action of SWI5 enables SBF to bind and activate the transcription of the HO gene
- SWI5 (SWItch 5) can recruit nucleosome modifier proteins SWI/SNF and histone acetyltransferases which act on nucleosomes over the SBF sites exposing it)
- SBF recruits Mediator complex and HO gene activated
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Signal Integration (Indirect #4):
Explain Cooperative Binding of Activators at the Human β-Interferon Gene
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Human β-Interferon Gene activated in cells upon viral infection. Infection triggers 3 activators:
1. NFkB (Nuclear Factor kappa of B cells),
2. IRF (Interferon Regulatory Factor)
3. jun/ATF (“ju-nanna”/A. Transcription Factor..)
- In addition to the activators listed above, another protein binds the enhancer- HMGA1 which unbends DNA and has AT hooks to bind to minor groove
- the activators bind highly cooperatively to enhancer (tightly packed) and form enhanceosome which recruits nucleosome remodellers and transcriptional machinery
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why are Eukaryotic regulators so diverse and complex
Eukaryotic regulators work on different enhancers and in many possible combinations
- Combinatorial Control At Heart of Complexity Diversity of Eukaryotes
- complex multicellular organisms have combinatorial control involving many more regulators & genes than shown
- Regulation can involve many of the same proteins working together
- both repressors and activators can be involved
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Eukaryotic repressors bind DNA and shut down gene transcription in what ways? (4)
Eukaryotes don’t use repressors to physically block RNA Polymerase from the promoter
1. Repressors can interfere with activators (block its binding site)
2. Repressors can lack activating regions but still bind another activator (occluding its activating region)
3. Repressors can bind DNA sites and interfere with parts of transcriptional machinery (direct repression)
4. Indirect repression-> Represssors can recruit nucleosome modifiers (histone methylases), compact chromatin, or remove groups recognized by the transcriptional machinery (histone deacetylases- remove acetyl groups from tails of histones)
Histone modification (methylation- adding methyl groups to histone tails) is a type of “gene silencing”
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Yeast transcriptionally repress Gal genes when preferred sugar - glucose - is present,
How does yeast know glucose is present so it knows when to shut off galactose catabolic genes?
In the presence of glucose, Mig1 binds and switches
off the Gal1 genes by binding between UASG and Gal1
-Mig1 recruits a “repressing complex” containing the Tup1 protein that in turn recruits histone deacetylase
-Histone deactylase activity causes nucleosomes to be repackaged, thus blocking transcription
-Mig1-Tup1 can also directly inhibit transcriptional machinery
->Blocks galactose metabolism when glucose present!
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describe Gene “Silencing” By Modification Of Histone & DNA
“Transcriptional silencing” or “gene silencing” is a position effect -> I.e., a gene is silenced because of where it is located, not in response to a specific environmental signal -> silencing can spread to other genes, even distant ones
- The most common form of silencing is associated with a dense form of chromatin called heterochromatin
- Histone proteins can be modified to disrupt gene transcription (deacetylation)
- Transcription can also be silenced by methylation of DNA by enzymes called DNA methylases -> blocks protein binding to DNA (Methylated DNA can also recruit repressors + histone deacetylases)
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how is Silencing in Yeast Mediated by Deacetylation and Methylation of Histones?
Yeast utilize a silencing complex that promotes gene silencing spread in DNA
- Telomeres & ribosomal genes are “silent”
- final 1-5 kb of each chromosome is a folded, dense structure
- Silent Information Regulators (SIRs), Sir2 is a histone deacetylase
1) Rap1 binds to the telomere
2) Rap1 recruits SIR2
3) Nonacetylated tails then binds SIR3,4 to silence those genes
* Methylation of Histone H3 tail blocks the Sir2 binding to prevent spread of silencing to undesired regions
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epigenetic regulation?
The inheritance of gene expression patterns, in the absence of either mutation or initiating signal
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explain how Epigentic regulation can silence genes over generations by methylating DNA
DNA methylation patterns can be maintained through cell division
- DNA methylation is reliably inherited, so-called maintenance methylases modify hemimethylated DNA
- Nucleosome and DNA modifications can provide the basis for epigenetic inheritance, repressor proteins can be passed along (e.g., λ..)
- Methylated nucleosomes in daughter molecules recruit proteins with chromodomains to methylate the adjacent nucleosomes
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how is Methylation a signal to proteins to completely shut down gene expression
- With no methylation, gene transcription only depends if RNA Pol, activators, etc. are present -> quickly reversible, leaky…
- Methylation of DNA serves as recruitment signal for proteins that bind methyl-DNA
- >These methyl-DNA binding proteins then recruit histone deacetylases & methylases, or chromatin remodeling complexes
- >This completely shuts down transcription and often forever to prevent inappropriate gene expression
nucleic acids
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