Translation

polyPhe

poly Lys

Synonym codons

...CCA, terminal 3'OH or 2'OH is aminoacylated

aminoacyl RNA synthetases

• amino acid: ATP in, pyrophosphate out
• enzyme.aminoacyladenylate: tRNA in, AMP out
• amino acid-tRNA

• 1 type of amino acid
• all cognate tRNAs

The second step: IleRS can charge Val with AMP, but hydrolyses valul-aminoacyladenylate when tRNA^ile is added

U, C or A

• 2/3 RNA
• 1/3 protein

• Complete: 70S
• Large subunit (peptide transferase): 50S
• Small subunit(mRNA): 30S

• Complete: 80S
• Large subunit (peptide transferase): 60S
• Small subunit(mRNA): 40S

• Mr
• particle shape
• Svedberg units

• Small subunit binds to mRNA
• Large subunit joins it at the initiating AUG codon

1 ribosome/80nt

• Bacteria: start with formyl-methionine
• Formyl rapidly removed, met may be removed more slowly
• formyl-Met-X cut by deformylase then aminopeptidase

• Eukaryotes: start with methionine (AUG)
• May be removed later depending on the next amino acid
• Met-X, bond broken by aminopeptidase

• Shine-Dalgarno sequence: 5'-GGAGG-3' nearer 5' end
• Recognised by CCUCC at the end of the 16S RNA in the 30S subunit
• Each cistron in polycistronic mRNA has its own AUG and Shine-Dalgarno sequence

• 'Scanning ribosome model'
• eIF4F (eukaryotic initiation factor) on 40S subunit binds to 5' cap
• scans along RNA towards 3' end (using ATP)
• Locks on at AUG, 60S subunit joins
• sometimes, different start sites can be used to generate different proteins from one mRNA

• 'Internal initiation of translation'
• first discovered in picornaviruses
• IRES (internal ribosomal entry site) in the 5'UTR directs the binding of the ribosome to the mRNA

dicistronic assay: put IRES between two coding genes; if 2nd one is expressed, then the IRES works

Hepatitis C

• P site: for peptidyl-tRNA
• A site: for aminoacyl-tRNA

• Charged aa-tRNA binds to A site, using EFTu (Elongation factor protein) (bac) or eEF-1 (euk)
• GTP hydrolysis causes the dissociation of EFTu from the ribosome
• A peptide bond forms from the peptide held by the tRNA already in the P site eg by peptidyl transferase
• The bond between the P site peptide and the P site tRNA is broken
• The ribosome moves 3 nucleotides along the mRNA (translocation) using EFG (bac) or eEF-2 (euk) and GTP
• The peptidyl-tRNA (now with its peptide joined to the peptide next door) moves into the P site, while the deacylated peptide (that WAS in the P site) is kicked out, perhaps through the E site

• EFTu (bac) and eEF-1 (euk) bind the aminoacyl-tRNA to the A site
• EFG (bac) and eEF-2 (euk) cause translocation of the ribosome

If a stop codon (UAA, UAG, UGA) is in the A site, RF (release factor) binds, meaning peptidyl transferase binds to polypeptide to H2O - hydrolysis of the peptidyl-tRNA and therefore release

• Diphtheria toxin: covalently modifies eEF2 - cell death
• Ricin: ribsome inactiated by removal of a single adenosine base from the eukaryotic 28S rRNA via its glycosidase activity

• Streptomycin: binds to 30S unit, inhibits initiation, or causes misreading of mRNA at low concentrations
• Tetracycline: inhibits binding of aminoacyl-tRNA to the 30S subunit
• Oxazolidines: inihbit formation of initiation Met_i-30S ribosomal subunit-mRNA complex
• Chloramphenicol: inhibits peptidyltransferase activity of 50S subunit (for typhoid and bacterial meningitis)
• Erythromycin: blocks progression of nascent peptide by binding to 50S subunit by the peptide exit tunnel

• Global: eg interferon response
• Specific: eg ferritin

• eIF2 is an initiation factor that binds Met-tRNA_i and GTP and delivers them to the 40S subunit
• eIF2's activity is REDUCED by PHOSPHORYLATION
• one eIF2 kinase is activated in response to dsRNA, which is usually a by-product of viral infection
• replication is inhibited

• Transferritin protects the cell from too much iron by binding it
• When [Fe] is low, IRP (iron regulatory protein) binds to IRE (iron response element), which is in a hairpin in the 5'UTR of the ferritin gene
• IRP binding to IRE means the 40S subunit cannot bind to the 5'cap
• -> Initiation of ferritin synthesis does not happen when [Fe] is low

• When miRNAs:
• Inhibit translation
• Degrade mRNA
• Degrade protein

• RNA sequences in 5'UTR/introns that bind small molecules to regulate transcription
• eg thiamine (B1) levels directly control thiamine synthesis

• Lysosomes
• Proteasomes

• Non-selective in well-nourished cells: degrade longlived proteins and organelles all the time
• Selective after prolonged fast
• Import and degrade proteins with specific pentapeptide Lys-Phe-Glu-Arg-Gln (KFERQ) - selectively lost from tissues that atrophy in response to fasting eg liver, kidney, not brain, testes
• Also macroautophagy

Mediate destruction of short lived and ubiquitinated proteins

• 1) E1 (ubiquitin activating enzyme) links C-terminal glycine in Ub to -SH in E1 forming a thioester bond (uses ATP)
• 2) E2 (ubiquitin conjugating enzyme) ligates Ub to itself
• 3) E3 (ubiquitin ligase) ligates Ub to the e-NH2 groups of lysines in the protein destined for destruction
• This is related to Cyclin B?

Ubiquitin peptidases

• 1) Recognition: Ubiquitinated proteins bind to proteins in cao
• 2) Dissociation: ATP dependent process unravels the protein and spits out the Ubs
• 3) Translocation: The protein is fed into the shaft - the inner two rings are responsible for proteolysis
• 4) Destruction: protein is entirely degraded into 8 amino acid peptides which are released

• N-end rule: 5 specific amino acids at N-terminus
• PEST sequences: Pro, GLu, Ser, Thr - phosphorylated on S or T
• D-box: seems to be a sequence required for cyclin ubiquitination