Gene Expression Flashcards
Transcription
DNA to RNA
Translation
RNA to protein
Initiation (transcription)
RNA polymerase binds to promoter
Elongation (transcription)
DNA helix unwinds and RNA polymerase adds nucleotides in the 5’ to 3’ direction
Termination (transcription)
Polymerase reaches terminator sequence
Processing (transcription)
Polyadenylation & splicing
Histidine decarboxylase
Enzyme responsible for the production of histamine
Diamine Oxidase
Terminates the action of histamine
Initiation and pre-initiation complex
- TF2D binds to the TATA sequence:
-This causes other transcriptional factors to bind
-This initiates the formation of the pre-initiation complex - The pre-initiation complex brings RNA pol II to the promoter region (Transcription initiation start site)
-PIC also denatures DNA (DNA has to unwind in order to use one strand as the template)
-The PIC then positions the DNA template to the RNA pol II active site
Why does RNA pol II need the pre-initiation complex?
RNA pol II does not hav enough energy on its own to start transcription, it needs the TF’s (on PIC) to help bind and unwind DNA
Why do we need capping in transcription?
The RNA transcript is single stranded. Thus, it is less stable so the 5’ cap is added to keep it from degradation
How capping works:
- The C terminus of RNA pol II is phosphorylated by kinases
- Once the phosphate groups are added, the capping enzymes come in and add the cap to the 5’ end of RNA
- Transcription can the proceed
Polyadenylation
- On the pre-mRNA there is a PolyA signal
- The PolyA signal is recognized by the CPSF enzyme
- CPSF binds to the signal and initiates the cleavage event of about 20-30 nucleotides downstream
- The cleavage event results in a new 3’ hydroxyl. From the 3’ hydroxyl, the PAP enzyme comes in and adds all the A’s. Once the tail reaches 200-300 A’s, the PAP disengages and now we have the Poly A tail.
(If the poly A tail reaches a certain length it signals the mRNA for degradation)
CPSF
Cleavage and polyadenylation specificity factor
PAP
Polyadenylate polymerase
Splicing
This is the last stage before leaving the nucleus
1. The SnRNPS’S BIND TO THE 3’ AND 5’ end of the intron
2. They cleave at the 5’ and the 3’
3. The Introns are looped out and the exons are joined together
SnRNP
the RNA of spliceosomes. They recognize the split junctions between the introns and the exons.
Initiation (Translation)
Binding to ribosome at the 5’ cap
Elongation (Translation)
tRNA add amino acids to the growing chain
Termination (Translation)
Ribosome reaches a stop codon
Processing (Translation)
(critical for cell signalling)
modification or addition of a functional group- glycosylation, acylation, methylation
Initiation (Ternary complex)
- The ternary complex binds to the 40s subunit
- Once the ternary complex binds to the 40s subunit, eIF4E binds to the 40s subunit. eIF4E binds to the 5’ cap. This is how the ribosome finds the mRNA.
The Ternary Complex
Is made up of GTP, EIF2 (INITIATION FACTOR), tRNA with methionine (starting amino acids)- translation cannot occur without the first amino acid
Initiation (scanning)
translation
-the ribosome binds to the 5’ end of mRNA
-right beside the 5’ end is the untranslated region (UTR)
-the complex scans through the UTR in order to find the start codon
-once at the start codon, a lot of the initial factors are released
-because of their release the 60s subunit can now join the 40s subunit
-scanning is an energy dependant process
Elongation (detailed) (translation)
- tRNAs go into the A site
- The tRNA then moves to the P-site. The is where the peptide bond is formed
- They then go into the E site and leave
Termination (detailed) (translation)
Ribosome eventually reaches a stop codon (eg; UAA, UAG, UGA) and falls off, since there is not anticodon to the stop codons
-release factors enter the A site and mediate the process (dissociation of mRNA from the ribosome)
Histones
Are proteins that provides structural support for a chromosome. Each chromosome contains a long molecule of DNA, which must fit into the cell nucleus. To do that, the DNA wraps around complexes of histone proteins, giving the chromosome a more compact shape.
Histone modification (up regulation)
Enzyme: Histone acetyltransferase (HAT)
What does it do: add acetyl groups
Histones have positively charged lysine tails. Acetyl groups are negatively charged. Once you add acetyl groups to the Histone, this mitigates the positive charge of the lysine tails. That way, DNA, which is negatively charged, is not as tightly bound to the Histone. Allows RNA pol II/ PIC to have better access to DNA, increasing rate of transcription
Histone modification (down regulation)
Enzyme: Histone deacetylases (HDAC)
What does it do: remove acetyl groups
Histones have positively charged lysine tails. Acetyl groups are negatively charged. Once you remove acetyls group from the HIstone, the DNA which is negatively charged can more tightly bound to the Histone. (Think of it like the acetyl groups steal the lysine tails from the DNA, so once you remove the tails, DNA can bound more tightly to the lysine tails). RNA pol II/ PIC have less access to DNA, decreasing rate of transcription
DNA METHYLATION (cytosine methylation)
Regulates gene expression by adding a methyl group to cytosine. This recruits proteins involved in gene repression or inhibits the binding of transcription factor(s) to DNA.
methylation blocks the PIC access to the TATA box, in the promoter region, which prevents transcription from starting
DNA methyltransferase (DMNT)
The enzyme that adds the methyl groups to cytosine nucleobases in CpG islands in the promoter region
DNA DEMETHYLATION
Regulates gene expression by removing the methyl group.
The removal of the methyl group from the cytosine in the CpG islands restores the PIC access to the TATA box and allows transcription to continue.
Activators
Bind to regions called enhancers. These stabilize PIC and thus increases rate of transcription. When the activator binds, it changes the structural format of DNA making the PIC more stable
Repressors
Bind to regions called silencers. These destabilize PIC and thus decrease the rate of transcription
Alternative Polyadenylation
Alternative sites which the polyA tail can be added.
Most mRNAS have different poly A signals. Depending on which signal is used, there are different 3’ ends the mRNA could have. This means that there is the possibility of
A: UTR region
B: No UTR region
If there is (A) UTR region:
-miRNA have complementary to this UTR region
-when miRNA binds to this region it can:
1. Induce mRNA degradation:
No mRNA = No template = No translation
2. Affect the export of the mature mRNA to cytoplasm:
No export = No translation
miRNA
Micro RNAs which are class of non-coding RNAs. They influence how well mRNAs are translated later on because miRNAs affect stability, structure and function of mRNA
Alternative splicing
snRNPs can cleave:
-Portions of exons out
-Can cleave exons out altogether
Evidently, this will affect the protein that’s formed downstream. Allows a single gene to code for multiple proteins.
Kinases
If Kinases don’t phosphorylate RNA pol 2, then the capping enzyme cannot come in and add the 5’ cap.
No cap
= No protection from being broken down
= eiF4e can’t bind to 5’ cap = 40 S cant find mRNA = no translation
Polysomes
Location: Cytoplasm
Possibility: not rare
Are clusters of ribosomes that translate mRNA at the same time. this parallel processing speeds up translation. Way more proteins can be formed because they are each forming their own polypeptide.
Phosphorylation of eIF2
Occurs during initiation of translation: This is the most common target.
Eif2 phosphorylation blocks the formation of ternary complex. This is true because when eiF2 is phosphorylated it can no longer act with eif2b. This completely stops translation from occurring.
Importance of eIF2
-When ternary complex reaches start codon, GTP is transferred to GDP.
-eif2B then swaps GDP to GTP, this regenerates the
ternary complex (you need it right away)
-eif2 phosphorylation = no interaction with eif2B (GEF)
= no regeneration = no ternary complex = NO translation
eIF2 falls under the umbrella of a guanine nucleotide-exchange factor (GEF)
Dephosphorylation of eIF2
allows the formation of the ternary complex to proceed (eIF2 can act with eIF2B)
Amino acids (translation) (stop codons that don’t stop)
Location: Cytoplasm
Possibility: rare
Pyrrolysine (Pyl) is encoded by UAG
-The polypeptide chain never actually stops growing even though UAG is a stop codon
Selenocysteine (Sec) is encoded by UGA
-When SELENIUM is NOT present, UGA does not work as a STOP codon, so the end result is a non functional-truncated protein
-Mercury Poisoning
Tetracycline (translation)
Location: Cytoplasm
Possibility: Not super rare, only applicable to bacterial illness, ex; could be used for h pylori bacteria
START
-Tetracycline binds on the small subunit (30S) at the A (Aminoacyl) site to prevent the tRNA from binding there
-Thus, this stops from translation from occurring
END
-If the Tetracycline cant compete enough with A-Site, it will persist and Inhibit release factors R1 and R2 from termination. This way protein synthesis will also be inhibited.
C Terminus (Processing)
When you add a lipid anchor to the C terminus, the protein can enter a membrane without having a transmembrane domain (when proteins have the transmembrane domain, this is all they needs to be anchored into the membrane)
N Terminus (processing)
N-linked glycosylation
-The addition of a sugar molecule to N terminus
-Proteins are now soluble and membrane bound
-Called Glycoproteins
-Glycoproteins can now travel to: Golgi
apparatus, lysosomes, plasma membrane, extracellular space
What happens if no N-Linked Glycosylation?
Some cancer drug, tunicamycin has an
EFFECT of reducing N-linked glycosylation which prevents some proteins from being transported to the golgi apparatus.
Temperature (Processing)
Cold temp effect; translation, eIF2 phosphorylation
Factors involved: ribosomes, mRNA
-When you test in the cold, they have a different value of total proteins. So it’s a generic percent of protein product.
-Ribosomes work different at different temps. At the colder temp, the ribosome can’t work at the same rate and works at a slower rate.
-Proteins are folded at a different rate at a colder temp, because the folding mechanism are working at a slower rate.
-Downregulation; change overall protein synthesis to save energy.
-Body temp decreases drastically; Cells are designed to work at a certain temp change. So, this slows down
protein production;
Viruses
Are infectious, obligate intracellular parasites composed of genetic material surrounded by a protein coat and/or an envelope derived from a host cell membrane. (has to get into host in order to survive)
Need to get into a cell to replicate and they MUST make mRNA that the host can translate. All viruses rely on the host protein synthesis machinery.
