Protein synthesis, modification and trafficking Flashcards
(41 cards)
1
Q
Briefly describe the flow of genetic information from the nucleus to the cytoplasm
A
- mRNA is synthesised from DNA in the nucleus
- mRNA travels into the cytoplasm via a nuclear pore
- Ribosome binds to mRNA and synthesises a polypeptide (protein) from the mRNA structure
2
Q
Why is protein synthesis an important process for a cell?
A
- Because proteins are vital for cell growth, proliferation and survival
3
Q
Why must protein synthesis be tightly regulated?
A
- Because it’s an expensive process for the cell (requires a lot of energy and uses up a lot of the cells resources)
4
Q
What about the process can protein synthesis regulatyion control?
A
- Can control overall rates of protein synthesis as well as the expression of specific transcripts
5
Q
What is protein synthesis inhibited by?
A
- It’s inhibited by cell stresses and withdrawal of nutrients e.g.
- Serum deprivation
- Temperature shock
- DNA damage
- Viral infection
6
Q
What are some of the differences between prokaryotic and eukarytotic mRNA?
A
-
Prokaryotic mRNA
- Polycistronic - more then one coding region
- Normally stable
- Transcription and translation can occur on same trnascript due to lack of nuclear membrane
-
Eukaryotic mRNA
- Monocistronic - only one coding region
- Capped and polyadenylated - increases stability
- 5’ and 3’ UTRs
7
Q
Briefly describe the structure of the 5’ Cap
A
- Structure is 7-methylguanosine
- Attached to the 5’ end of the mRNA by a 5’ to 5’ triphosphate bridge
8
Q
What are the functions of the 5’ cap?
A
- Seals end of mRNA protecting it form nuclease digestion
- Potent initiator of protein synthesis - binding site for eIF4E
9
Q
Briefly explain the process of 5’ capping
A
- Removal of 5’ terminal phosphate by the enzyme triphosphatase
- Addition of 5’ terminal GMP by enzyme guanylyl transferase
- Methylation of guanine base at position 7 by enzyme guanine-7-methyltransferase
- Methylation of ribose (in some cases)
10
Q
What are the functions of the poly(A) tail?
A
- Protects mRNA from enzymatic degradation
- Aids in transcription termination, export of mRNA from nucleus and translation
11
Q
Briefly describe the process of polyadenylation
A
- Recognition of the AUAAA sequence (polyadenylation signal) by specificity componenets
- CPSF (Cleavage and polyadenylation specificty factor) then cleaves AUAAA sequence
- Initial poly(A) polymerisation by poly (A) polymerase - addition of multiple adenosone monophosphates
- This is followed by binding of poly(A) binding protein (PABP)
- More poly(A) polymerisation and binding of more PABP
12
Q
Describe the structure of tRNA
A
- Single RNA strand of about 80 nucleotides
- G-C rich - allows tRNA to fold back on itself as there are regions of base pairing between the Gs and Cs which form loops
- Anticodon loop recognises codons on mRNA
- Loops seperated by stems - acceptor stem is where charged amino acids are addded
13
Q
What is the function of the enzyme Aminoacyl tRNA synthetase?
A
- Links an amino acid to 3’ end of acceptor arm to produce an aminoacyl-tRNA
14
Q
Describe some characteristics of the 80S ribsosome
A
- Consists of large and smal subunits - each consist of 50% protein and 50% RNA by mass
- Small and large subunits bind together during initiation
- Translation takes place in the cavity between the two subunits
- Has 3 binding sites for tRNA:
- E (Exit site)
- A (Aminoacyl-tRNA binding site)
- P (Peptidyl-tRNA binding site)
15
Q
What enzymatic activity is associated with the 60S (large subunit)?
A
- Peptidyl transferase activity
16
Q
Why must translation occur at the correct speed?
A
- Translation must go fast enough to supply protein but slow enough to avoid too many errors
17
Q
What are the 3 stages of CAP-dependent protein synthesis?
A
-
Initiation
- 40S ribosomal subunit and initator tRNA bind
- Recognition of 5’ CAP
- scanning to locate start codon (AUG)
- Formation and binding of ternary complex to 60S ribosomal subunit
-
Elongation
- tRNA brings amino acids to the ribosome in the order specified by codons on mRNA
- Ribosome catalyses peptide bond formation between the amino group of each amino acid
-
Termination
- At 3’ end cof coding sequence ribosome encounters stop codon
- Polypeptide chain is released
18
Q
Why is initiation the rate-limiting step in CAP-dependent protein synthesis?
A
- Initiation requires solube eIFs (eukaryotic initiation factors) to occur
- Initiation is rate-limiting step because eIFs are in short supply within cytoplasm
19
Q
What are the 3 main stages of Initiation?
A
- Assembly of the 43S pre-initiation complex
- Binding of mRNA to the 43S complex
- Assembly of 80S initiation complex
20
Q
Describe the process of the assembly of the 43S pre-initiation complex
A
- Met-tRNA associates with eIF2-GTP to form the ternary complex
- 40S ribosomal subunit is recognised by eIF3
- eIF3 binds to and traps the 40S ribosomal sunbunit to form the 43S ribosomal subunit
- Ternary complex associates with the 43 ribsosomal subunit to form the 43S pre-initiation complex
21
Q
Describe the structure of eIF2
A
- α subunit - Phosphorylation site found here
- β subunit - K boxes (involved in interaction between eIF2B adn eIF5) found here
- γ subunit - GTP binding site found here
22
Q
Describe the process of the binding of mRNA to the 43S pre-initiation complex
A
- eIF4 recognises and binds to 5’ CAP on mRNA
- eIF4G binds to eIF4E
- eIF4G has a binding site for eIF4A allowing it to bind
- eIF4A unwinds any secondary structures within the 5’ CAP of the mRNA
- eIF3 also binds to eIF4G
- eIF3 allows for the 43S pre-initiation complex to bind to eIF4G
- This results in the formation of the 48S pre-initiation complex
23
Q
How does eIF4G act as a scaffold molecule?
A
- eIF4G has multiple binding sites with other initiation factors such as:
- PABP - poly(A) binding protein
- eIF4A
- eIF4E
- eIF3
- It acts as a scaffold molecule has all these binding sits allow it to bring together several other translation initiation factors
24
Q
What does the fact that eIF4G can bind to the PABP mean for mRNA?
A
- Fact that eIF4G can bind to PABP on 3’ end of mRNA means that the mRNA can be circularised
25
Whaat other initiation factor can bind PABP and can therfore cause circularisation of mRNA?
* eIF4B - also part of 43S pre-initiation complex
26
What happens after the mRNA binds to the 43S pre-initation complex?
* Ribosomal scanning occurs which allows met-tRNA to recognise start codon AUG
27
Describe the process of ribosomal scanning to find AUG start codon
* eIF4A helps unwind secondary structures (stem loops) at 5' end of mRNA
* This helps 48S pre-initiation complex to scan along mRNA smoothly and locate AUG
28
How does the structure/position of the secondary structures (stem loops) effect how mRNA is translated?
* Secondary structure near the 5’ end impedes the attachment of the 43S pre-initiation complex to the mRNA and/or reduces the ability of the complex to scan along the mRNA
29
How does CAP-independent translation allow for more efficient attachment of the 43S pre-initiation complex and loaction of AUG compared to CAP-dependent translation?
* mRNAs that can be translated using CAP-independent translation have an internal ribosome entry site (IRESs)
* These allow direct binding of the 43S complex to the mRNA without the need for cap recognition or scanning
30
Explain the process of the assembly of the 80S initiation complex
1. eIF5 catalyses the binding of 60S ribsosomal sbunit to the 48S pre-initiation complex to form 80S ribosome by hydrolysing GTP to form GDP
2. GDP binds to eIF2 to form eIF2-GDP
3. met-tRNA occupies the P site of the 80S ribosome
31
Describe the process of the formation of the first peptide bond and elongation
1. 80S ribosome assembled and met-tRNA occupies P site - this leaves unocccupied A (Aminoacyl-tRNA binding) site next to it
2. Aminoacyl-tRNA binds to A site on 80S ribosome bringing with it correct charged amino acid - catalysed by eEF1 (eukaryotic elongation factor 1) and requires GTP
3. Peptidyl transferase activity of 60S subunit will catalyse formation of peptide bond between first 2 amino acids
4. Amino acid bound by aminoacyl-tRNA is released and ribosome moves along to next codon on mRNA - this step is called translocation and is catalysed by eEF2 and requires GTP
32
Describe the process of the termination of the polypeptide chain
1. Elonagtion continues until the Aminoacyl-tRNA binding site occupies a STOP codon - e.g. UAA, UAG, UGA
2. The STOP codon is recognised by and bound to by a release factor called eRF
3. This causes hydrolysis of the last peptidyl-tRNA bond and release of the completed polypeptide chain
4. Dissociation of the two ribosomal subunits from the mRNA
33
What is a polysome?
* A structure that consists of multiple ribosomes attached to a single mRNA
34
Give an example of how translation is a very efficient process
* Ribosomes are constantly recycled for further rounds of translation of the same transcript
35
What is the difference between proteins synthesised by free ribosomes and those synthesised by ER-bound ribosomes?
* Free ribosomes -Synthesize soluble proteins that function in the cytosol
* ER bound ribosomes – Synthesize proteins destined either for incorporation into cell membranes or for export from the cell
36
Can a ribosome switch from being free to being bound to the ER and vice versa?
* Yes ribosomes can switch back-and-forth between the two types
37
What are the 2 types of regulatory control of translation?
* **Global regulation of transcription**
* Usually by modification of translation initiation factors e.g. eIF2, 4E-BP1
* Achieved by changes in the phosphorylation state of these factors or regulators that interact with them
* **mRNA specific regulation**
* **U**ses elements in the 5’ and 3’ untranslated regions
38
Explain how 4E-BP1 regulates protein synthesis
* In cells that are active and unstressed 4E-BP1 is highly phosphorylated and inactive
* However, if a cell becomes stressed it becomes de-phosphorylated
* When this occurs 4E-BP1 will ibhibit protein synthesis
39
Explain how eIF2 regulates protein synthesis
* eIF2 is normally de-phosphorylated and therefore active
* If a cell becomes stressed eIF2 becomes phosphorylated on its α subunit
* This causes the inhibition of protein synsthesis
40
Explain how eIF4G regulates protein synthesis
* When eIF4G gets cleaved by caspase 3 or becomes degraded it inhibits protein synthesis
41
eIF4E (oncogene) is often overexpressed in tumour cells. Why is this?
* Because in tumour cells you have a population of highly structured mRNA molecules
* This means when eIF4E binds to the 5' CAP it recruits eIF4G and eIF4A which is needed to unwind those secondary structures on the mRNA
