Protein Synthesis Flashcards
Why are proteins important?
Crucial for cell growth, proliferation and survival
How is protein synthesis regulated?
- Expensive process for the cell so is tightly regulated
- regulation can either control overall rates of synthesis throughout the cell or modulate specific reactions
What inhibits protein synthesis?
- cell stresses
- withdrawal of nutrients
- serum deprivation
- temperature shock
- DNA damage
- viral infection
- hypoxia
What does a prokaryotic mRNA contain?
- polycistronic = more than one coding region
- messages given are fairly unstable
- absence of nuclear membrane leads to transcription/translation occurring on the same transcript
What does a eukaryotic mRNA contain?
- usually monocistronic (one coding region)
- capped and polyadenylated so quite stable
- 5’ to 3’ untranslated regions (UTR)
What is the CAP structure?
- found on eukaryotic mRNAs
- at the 5’ end
- seals end of mRNA protecting it from nuclease digestion
- landing pad for eIF4E (cap binding protein)
What is polyadenylation?
- poly A tail found at the 3’ end
- protects mRNA from enzymatic degradation
- aids in transcription termination and exportation of mRNA from the nucleus into cytoplasm
What are the stages in 3’ polyadenylation of newly synthesised mRNAs?
- recognition of AUAAA sequence by specificity components which is cleaved by cleavage factors
- undergoes initial poly(A) polymerisation by poly(A) polymerase
- binds to poly(A) binding protein (PABP)
What are general characteristics of tRNA molecules?
- single RNA strand of approx 80 nucleotides
- helps decode mRNA sequence into a protein
- functions at a specific site in the ribosome during translation
- aminoacyl tRNA synthase links amino acid to 3’ end of acceptor arm to produce an aminoacyl-tRNA
What is the 80S ribosome?
- consists of large (60S) and small (40S) subunits
- each subunit contains approx 50% proteins and 50% rRNAs (by mass)
- large and small subunits bind together during initiation
- translation takes place in the cavity between the two subunits
- peptidyl transferase activity is associated with the large 60S subunit
- has three binding sites = E, P, A
What are some facts about protein synthesis?
- translation must go fast enough to supply protein but slow enough to avoid too many errors
- error rate is 1 in 10^4 incorrect amino acid
- ribosomes add 20 amino acids per second to a polypeptide chain eg the synthesis of actin 375 AAs takes 20 seconds
- it is energetically expensive
What are the 3 stages of CAP-dependant protein synthesis?
- Initiation:
- small ribosomal unit and initiator tRNA bind
- CAP is recognised and the start AUG codon is scanned
- ternary complex binds large ribosomal unit - 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
- more than one ribosome is active on any one mRNA (polysome) - Termination:
- at 3’ end of coding sequence, the ribosome encounters a stop codon
- polypeptide chain is released
What is the function of the initiation step of protein synthesis?
- initiation of translation is the rate limiting step and is tightly regulated
- more than ten soluble eukaryotic initiation factors (eIFs)
- aim is to bring mRNA to ribosome and initiator tRNA to AUG start codon
- the process is:
1. Assemble of the 43S pre-initiation complex
2. Binding of mRNA to the 43S complex
3. Assembly of the 80S initiation complex
What happens during the first step of initiation?
- assembly of the 43S pre-initiation complex
- association met-RnA with eIF2 to form ternary complex
- 40S is trapped as a monomer to form the 43S pre-initiation complex
What is the structure of eIF2?
- polypeptide chain initiation factor eIF2
- made up of three subunits:
1. gamma subunit = GTP binding site
2. alpha subunit = phosphorylation site
3. beta subunit = K boxes (involved in interaction of eIF2B and eIF5)
What happens during the second step of initiation?
- binding of mRNA to the 43S complex
- eIF4E binds to the CAP on mRNA
- eIF4G binds to eIF4E
- eIF4A unwinds mRNA secondary structure
- 43S preinitiation complex binds to eIF4G to form 48S preinitiation complex
What are the roles of different eIFs?
- eIF4E = recognition of 5’ cap on mRNA
- eIF4G = binds to eIF4E and eIF3
- eIF3 = acts as a bridge between eIF4G and the 40S ribosomal subunit, hence required for mRNA binding to the ribosome
What does the 43S pre initiation complex also interact with?
- interacts with the 3’ end of the mRNA is the poly(A) tail
- it is achieved by virtue of the ability of the poly(A) binding protein (PABP) to bind to bot eIF4G and another initiation factor eIF4B
- it results in the circularisation of the mRNA by association of the 5’ end with the 3’ end
What is the effect of secondary structure in the 5’ UTR of an mRNA
- 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
What is the function of internal ribosome entry sites (IRESs)?
- some mRNAs overcome the secondary structure problem by having complex internal ribosome entry sites (IRESs)
- they allow direct binding of the 43S complex to the mRNA without the need for cap recognition or scanning
- IRESs can replace the function of some of the initiation factors
- some viral mRNAs use IRES mediated CAP-independent translation, eg picornavirus
What happens during the third step of initiation?
- assembly of the 80S initiation complex
- the 40S/43S pre initiation complex scans to AUG start codon
- MET tRNA occupies the P site on the ribosome
- 60S subunit associated to form 80S complex
Describe the 80S initiation assembly?
- it involves:
1. Binding of the 60 ribosomal unit to the 43S.mRNA complex
2. Hydrolysis of the GTP bound to eIF2
3. Release of most of the initiation factors from the ribosome - these events require the involvement of yet another initiation factor eIF5 (has GTPase activity)
What are the stages of elongation?
- AA-tRNA binding - catalysed by eEF1, requires GTP
- Peptide bond formation - catalysed by ribosome itself
- Translocation - catalysed by eEF2, requires GTP
What are the stages of termination?
- Recognition of a termination STOP codon by a release factor (eRF) - eg UAA, UAG, UGA
- Hydrolysis of the last peptidyl-tRNA bond and release of the completed polypeptide chain
- dissociation of the 2 ribosomal subunits from the mRNA
What is the ribosome-polysome cycle?
- a polysome is a structure that consists of multiple ribosomes attached to a single mRNA
- ribosomes are recycled for further rounds of translation
Why are some ribosomes bound to the ER?
- free ribosomes synthesise soluble proteins that function in the cytosol
- ER bound ribosomes synthesise proteins destined either for incorporation into the cell membrane or for export from the cell
- ribosomes can switch between the two types
What are molecular mechanisms of translational control?
Global regulation of translation:
- usually by modification of translation initiation factors
- achieved by changes in the phosphorylation state of these factors or regulators that interact with them
mRNA specific regulation:
- uses elements in the 5’ to 3’ UTRs
What are some regulatory factors that inhibit protein synthesis?
- dephosphorylation of 4E-BP1 (tumour suppressor protein)
- eIF4G cleavage by caspase 3
- phosphorylation of eIF2 into eIF2aP
- overexpression of eIF4E (oncogene) leads to loss of cellular growth control
