Regulation of translation Flashcards
Describe the detailed structure of a processed mRNA and its importance in regulation of the mRNA.
The processed mRNA includes the coding regions, or a ORF, that encodes for the protein. It starts with a 5’ cap, which consists of a 7-methyl guanosine at its 5’ end. There is also a 5’ and a 3’ UTR (untranslated region) that is thought to be important in the efficiency of translation. A lot of essential mRNAs have a long 5’ UTR. It is thought that the longer the UTR, the more efficient the translation of the mRNA. The 5’ UTR includes a hairpin loop and the loop structures of IRES. These structures have to be unfolded for the protein synthesis to be initiated. The 3’ end also has a poly(A) tail. The 7-methyl guanosine and polyA tail play a role in mRNA stability.
What is the role of aminoacyl-tRNA synthetase in translation?
Aminoacyl-tRNA synthetase is able to recognise the correct set of tRNAs for a particular amino acid and brings them together in order to load the amino acid onto the tRNA. Therefore, it synthesises aminoacyl-tRNA. It does this by first binding ATP and the amino acid (forms aminoacyl-adenylate), which then binds to the D arm of the tRNA. The amino acid is then added to the 3’ end of this chain and ATP is hydrolysed to release energy and ADP. When the amino acid is in the complex with tRNA, it is then activated and able to participate in peptide bond formation.
Describe the cap dependent mechanism for the initiation of translation in eukaryotes.
The most common process of initiation is the cap-dependent initiation - it relies on the 5’ cap on the mRNA. eIF is an eukaryotic initiation factor, and eIF4F proteins are the specific ones that help with finding the initiation sequence on mRNA. eIF4E binds to the 5’ cap of the mRNA. It then recruits multiple other eIF proteins - eIF4A, eIF4B an eIF4G. These then unfold the 5’ UTR and scans the sequence from the 5’ to 3’ direction searching for the start codon.
Another protein PABP will bind to the polyA tail, and eIF4G will then bind to PABP. This leads to the circularisation of the mRNA, which is important for increasing the local concentration of initiation factors and ensures the translation of only intact mRNA.
Along with this, there is the formation of the 43S complex, a ribonucleoprotein complex. This includes more eIF proteins, such as eIF5, eIF3, eIF2, eIF1A and eIF1, as well as the 40S ribosomal subunit. eIF2 is bound to the first Met tRNA. eIF1A and eIF3 are important for promoting the binding of the eIF complex to the 40S subunit. The eIF4:mRNA complex binds to the 43S complex. This will then recruit the 60S subunit to form the full 80S ribosome. Once all the subunits are together, then initiation of the scanning of the mRNA for the start codon begins. The UTR may contain many AUG sequences, but there will only be one start codon - the correct AUG will be surrounded by the rest of the Kozak sequence, which will be needed to start translation.
Describe the cap independent, or Internal ribosome entry site (IRES), mechanism for initiation of translation in eukaryotes.
This mechanism involves specific IRES complexes that are within the mRNA, upstream of the start codon (usually in the 5’ UTR). This mechanism is usually used when the common pathway is blocked, in order to maintain protein expression and aid the survival of the cell. It is still unclear the mechanism that is used in eukaryotic cells.
Explain the role of eEF1A/B and eEF2 in the elongation phase of eukaryotic translation.
tRNA binds to EF-1A (elongation factor 1A) and this then guides the tRNA into the A site of the 80S. This process requires the hydrolysis of GTP. This is also mediated by EF-1B.
EF-2 also is bound to GTP and mediates the translocation of the polypeptide chain. Once the new tRNA enters the A site, the new amino acid must be added to the growing chain. This is mediated by the ribosomal subunits. But the translocation of the chain now from the A site back into the P site is mediated by the EF-2. The cycle then repeats again.
How is the correct sequence ensured by the ‘proofreading’ of translation?
The ribosome needs to ensure that the sequence of amino acids is correct. This is mediated by the affinity of the codon and anticodon sequence. Dissociation of the EF:tRNA:GTP complex occurs at a lower rate when the base-pairing between the codon and anticodon is perfect, and at a higher rate when they are not. This means that the affinity of the wrong anticodon is weak and dissociates before the hydrolysis of GTP.
Describe the mechanism of termination of translation in eukaryotes by the mimicry of tRNA by releasing factors.
eRF1 is structurally similar to tRNA and mimics tRNA by moving into the ribosomal A site, when it recognises a stop codon. Then, it catalyses a nucleophilic attack on the ester bond between the peptide and the P-site tRNA. This catalytic activity is stimulated by the GTP bound eRF3.
Briefly explain the importance of protein homeostasis, or proteostasis.
Protein synthesis is one of the most energy-dependent processes in the cell, so it is important to tightly regulate this. Processes such as translation regulation, folding, post-translational modifications and degradation are all involved in keeping the balance of proteins inside a cell. Dysregulation of this typically can result in pathological changes and disease. Proteins are therefore produced when the cell requires them, produced locally, or produced in a specialised manner e.g. IgG.
Describe the two different complexes that can be formed by mTOR in the presence of different signals, and what these complexes then induce within cells.
mTOR is able to respond to cellular conditions and regulate protein synthesis. It sense nutrient availability, energy levels, and mitogens (induce cell division). mTOR stands for mammalian Target fOr Rapamycin.
One complex that mTOR can form is in response to nutrient levels and energy levels in the cell - mTOR complex 1. This complex includes mTOR, Raptor, GbL and Rheb. mTOR acts as a kinase enzyme and Raptor is the substrate-presenting protein that brings the substrates to the mTOR to be phosphorylated. This process requires ATP. The two main substrates that are phosphorylated by mTORC1 are 4E-BP1 (4P) and S6K (P). These proteins are essential for cell growth, protein synthesis and metabolism.
mTOR complex 2 is activated in response to hormones and growth factors. It has a different substrate-presenting proteins, termed Rictor. It also includes GbL within its complex. mTORC2 goes on to phosphorylate PKB, which is essential for rearrangement of the cytoskeleton and cell proliferation.
What role does mTOR complex 1 play in the initiation of translation in eukaryotes?
mTORC1 can phosphorylate 4E-BP1 (eIF4E-binding protein 1), but only when there is sufficient nutrients, energy and growth factors present in the cell. Phosphorylation of 4E-BP1 prevents it from binding to eIF4E. This means eIF4E can then go on to play its role in initiation of transcription, and therefore increasing protein synthesis.
What is the relationship between mTOR and rapamycine?
Rapamycin is an immunosuppressant and acts as an inhibitor for mTOR.
What are some molecules that can lead to the inactivation of mTOR?
An increase in AMP concentration triggers activation of AMPK (AMP kinase), by phosphorylation from LKB (liver kinase B1). This activates TSC1 and TSC2, which are tumour suppressor complexes. TSC2 then goes on to inactivate mTOR, which in turn decreases the level of protein synthesis in the cell.
P13K/Akt/mTOR pathway is an intracellular signalling pathway that can be inhibited by PTEN. PTEN is a phosphatase, which can dephosphorylate the product of P13K. This product is essential for the synthesis of Akt. If Akt is not available, it cannot activate mTOR.
Explain how the cell can induce protein synthesis without mTOR in the presence of mitogens or under stress.
Extracellular signals, such as mitogens, can initiate protein synthesis because they bind to MAPK (mitogen-activating protein kinase) receptors, which sets off a cascade of proteins that lead to translational initiation. For example, ERK1/2 (kinase) can phosphorylate and activate MINK. This MINK will then go on to phosphorylate eIF4E, needed to initiate translation.
Intracellular stress signals can also set off cascades that activate MINK.
Explain the mechanisms of steric blockage and sequestration in regulating initiation complex assembly for translation.
Steric blockage involves the IRE sequence in the 5’ UTR, which forms a stem loop in the mRNA. IRP (iron regulating protein) can bind to this loop structure. This blocks the initiation complex from being able to move down the mRNA in search for the start codon. Therefore, inhibiting translation.
Sequestration of eIF4E is achieved by the binding of maskin to eIF4E, which inhibits it even if it has already bound to the 5’ cap. CPEB is also bound to maskin, and CPEB goes on to bind to CPE sequence in the 3’ UTR, which circularises the mRNA and sequesters translation.
Describe the regulation of cyclin B1 translation during the cell cycle.
Cyclin B1 is a regulatory protein involved in mitosis. The translation of cyclin B1 is induced during the M phase by the dislodgement of maskin/CPEB complex. As mitosis progresses, the polyA tail of the mRNA grows. This recruits more and more PABP (polyA binding protein) onto the polyA tail. PABP then mediates the dislodgement of maskin and therefore, initiates the translation of cyclin B1. Once the cell enters the S phase, the maskin/CPEB complex rebinds to sequester the translation of cyclin B1.
