11- Translation(●'◡'●)

Key ingredients for translation and what the purpose is

purpose: Production of protein from mRNA template

• ribosomes

• tRNAs

• aas (aa and tRNA= aminoacyl tRNAs)

• transcript mRNA

What’s a polysome (polyribosome)

• multiple ribosomes translating single mRNA at same time

• Occurs when gene transcriptionally active

Ribosome purpose, location and general structure

• Site of translation

• Found in multiple locations in cell

  • cytoplasm, ER membrane, outer nuclear envelope, MT matrix, chloroplast stroma

• Composed of 2 subunits (made of rRNA and proteins)

  • large (LSU) and small (SSU)

Ribosome composed of which rRNA (and proteins) in proks vs euks

• rRNA folding pattern highly conserved in all organisms

• Euks: 28S, 5.8S, 5S, 18S (expansions at surface)

• Proks: 23S, 5S, 16S

THESE ARE rRNA inside the ribosome that build each subunit

Famous rRNA features-LSU

• (23S/28S)

• Interacts w/ tRNA

• contains Peptidyl transferase center (PTC)=ribozyme= catalytic core

• Catalyzes peptide bond formation (ribozymal activity)

• a-sarcin-ricin loop (a seq)= highly conserved, target for toxins (23/28S)

• Catalytic core is a highly conserved ribozyme = LUCA

Famous rRNA fts-SSU

• SSU rRNAs= decoding center (interact w/ mRNA)

  • 3’ end of 16S bp(anti-SD) with Shine Dalgarno seq

  • 16S barcode= microbiome studies

  • 18S interacts with mRNA (IRES, Kozak maybe)

LSU proteins and SSU proteins

• LSU→ RPLs

• SSU→ RPSs

both RP but then L for large, S for small

Compare prokaryotic vs eukaryotic ribosomes.

Prokaryotes	Eukaryotes
30S + 50S = 70S	40S + 60S = 80S
Shine-Dalgarno	Kozak sequence
fMet initiator	Met initiator
Fewer protein factors	MANY eIFs

Organism	Small subunit	Large subunit	Whole ribosome
Prokaryote	30S (16S)	50S (23S + 5S)	70S
Eukaryote	40S (18S)	60S (28S + 5.8S + 5S)	80S

Ribosome 4 key sites

• mRNA binding site (prokaryotes only)

• E (exit site)

• P (peptidyl site)

• A (aminoacyl site)

• Roles

  • Scaffold

  • Proofreading

  • Catalytic activity

tRNA will

• bring amino acids to ribosome

  • each t RNA binds 1 specific amino acid

    • ester bonded to 3’ adenine

• Recognizes and binds to 1 or more codon sites, has anticodon

Anticodon

• 3 base seq on tRNA that bp with mRNA codons via H bonding

• anticodon forms a complementary RNA duplex with mRNA codon

Why can the anticodon in tRNA bind with more than 1 mRNA codon

• Flexibility in 3rd position of codon

• The wobble hypothesis

  • 3rd position wobble effect where.. in the anticodon

    • G can pair w/ C or U

    • U can pair w/ A or G

    • I can pair w/ U, C or A

Naming convention for anticodon

• 3’-5’

• mRNA 5’-GCC’3

• tRNA written 3’-CGG5’ → antiparallel bp

Aminoacyl-tRNA synthetases

• Enzymes that attach appropriate aa to correct tRNA

  • amino acid activation

• Cells have 20 diff tRNA synthetases

• “super specificity”= synthetases recognizing tRNAs involves

  • Anticodon loop

  • 3’ end

  • acceptor stem

What energy is required to add amino acid to tRNA?

• ATP→ AMP (hydrolyzed) this is catalyzed by aminoacyl tRNA synthetases

• “high energy bond” formed bw tRNA and aa

  • aminoacyl tRNA aka charged tRNA

Be able to draw mature mRNA for proks and euks

Differences bw proks and euks

Prokaryotes	Eukaryotes
Direct recruitment of ribosome to AUG start codon	Cap-dependent recruitment followed by scanning for AUG start codon
Shine–Dalgarno sequence in mRNA base pairing with anti-SD in 16S rRNA	Kozak sequence cap-dependent recruitment gets the ribosome on the mRNA, scanning finds the codon, and the Kozak sequence ensures the correct codon is chosen.
Special formylated tRNA (fMet) initiator	Methionine (Met) binds to AUG start codon
Much of what we know about termination is based on prokaryotes	Much of what we know in eukaryotes is based on yeast
—	Nucleus — transcription and translation are spatially and temporally separated
—	WAY more protein factors (trans) and mRNA structural features (cis) regulate the process

translation is primarily regulated at the initiation step. Why do you think that is?

• Bc its extreme energy expensive so cells avoid wasting resources by controlling intitiation

What helps ribosome find start codon in proks vs euks

Proks

• Shine dalgarno seq

• Base pairs w/ anti-SD in 16S

Euks

• Kozak seq

• Cap-dependent scanning

Bacterial translation initiation regulated through

• coordinated action of initiation factors

  • IFs

Step 1 of bacterial initiation

• Initiation factors (trans) IF1, IF2, IF3 bind to SSU

• GTP binds IF2

• IF3 blocks 50S joining (LSU)

  • roadblock

Step 2 bac intiation

• tRNA and mRNA recruitment

• fmet tRNA binds P site

  • IF2 assists specificity

• Shine-Dalgarno on mRNA

  • binds anti-SD in 16S rRNA (which is part of 30S subunit)

  • Start codon is now positioned correctly

• IF3 dissociates= roadblock lifted

Step 3 bac initiation

• LSU recruitment

• After IF3 released

  • 50S binds→ 70S initiation complex formed

• GTP hydrolysis releases IF1 and IF2 during complex formation

Initiation in euks regultaed through

• coordinated action of euk initiation factors

• eIFs

5 main events that initiate translation in euks

1. Initiatior met-tRNAi met recruitment= ternary complex formation

2. mRNA activation= cap binding eIF4F complex + PABP

3. formation of preinitiation complex (PIC)= tRNA + 40S and bunch of eIFs

4. recruitment of mRNA to PIC + scanning for AUG start codon

5. recruitment of 60S= licensed to translate

Ternary complex formation

• eIF2-GTP bind to Met-tRNAi Met

• ternary bc there’s 3 items : EIF2, GTP, Met-tRNAiMet

mRNA activation

• eIF4F complex binds 5’m7G cap

• PolyA binding protein (PABP) binds 3’polyA tail

• eIF4F complex binds PABP

  • circularized mRNA bc now a loop

Formation of 43S PIC

• eIF3, eIF1, eIF1A binds to 40S

• ternary complex joins party

• eIF5 jumps on too

• roadblocks

  • eIF1 sits in P site

  • eIF2-GTP blocks 60S binding (prevents it coming in too early)

Recruitment of mRNA to PIC= formation of 48S

• interactions bw PIC + eIF4F brings mRNA → 40S

• ATP dependent scanning for AUG start codon

  • correct AUG det by Kozak Seq

  • triggers removal of eIF1→ roadblock removed that was blocking P site

• Met-tRNAiMet moves into P site

Recruitment of 60S to complete initiation

• 60S and eIF5B recruited

• eIF5B GTPase activity promotes eIF2-GTP→ eIF2-GDP + Pi

• Releases eIF2→ roadblock removed

• 60S can now bind

• 80S formed and licensed to translate

Non canonical translation- IRESs (cap independent)

• IRESs= internal ribsome entry sites

• highly struc RNA seq that can recruit ribosome directly in cap-independent manner (ribs can bind mRNA without cap)

  • might still need some initiation factors

• Found in some viral RNA, and the struc of RNA can mimic tRNA

  • this tRNA like structure helps recruit the ribosome to the mRNA.

    • initiates translation at non-AUG start codon

• During viral infection, host cells often shut down normal cap-dependent translation.

• Viral RNAs with IRES elements can still recruit ribosomes, allowing viral proteins to be produced even when host translation is inhibited.

Elongation proks

• Repetitive 3 steps coordinated by 3 elongation factors (EF-Tu, EF-Ts, EF-G)

1. Binding of aminoacyl tRNA

• Specified tRNA enters A site

2. Peptide bond formation

• AA on tRNA in P site hydrolyzed + peptide bonded to aa on A site tRNA

• this catalyzed by ribozyme site in 23/28S rRNA

3. Translocation

• Motion of ribosomal subunits moves the complex 3 nucleotides on mRNA, so tRNA in P site to enter E site, A site tRNA→ P site

Proks elongation Step 1-Aminoacyl tRNA binding

• Begins with AUG start codon in P site

• binding of tRNA to A site requires EFs

1. EF-Tu (GTPase turns GTP into GDP) binds GTP + then aa-tRNA^aa

2. EF-Tu-GTP-aa-tRNAaa binds to mRNA

3. GTP hydrolysis releases EF-Tu-GDP

4. EF-Ts (GEF) recycles EF-Tu

• GDP→ GTP

Prok Elongation Step 2-Peptide bond formation

• AA in P site cleaved from connection at 3’ end of tRNA

• AAs in P and A site located in peptidyl transferase center (PTC=catalytic core of the ribosome and acts as a ribozyme)

• covalent peptide bond formed bw aa on a tRNA in A site + aa on a tRNA in P site

Elongation step 3-translocation

• EF-G-GTP binds to 50S (LSU)

  • Ef-G= G protein

• GTP hydrolysis causes conformation change in ribosome, moving it 3 nucleotides along mRNA

  • • EF-G-GTP →EF-G-GDP + Pi

  • everything shifts over 1 site

• “A” site now free to receive next aa-tRNA

REPEAT till stop codon reached

Elongation is the same for proks and euks but switch

Prokaryotes Eukaryotes

EF-Tu eEF1A

EF-Ts eEF1B

EF-G eEF2

Termination proks

• Mediates through release factor (RF) which mimics a tRNA

• RF-GTP binds stop codon in A site

• Hydrolysis of RF-GTP→ RF-GDP + Pi

  • releases polypeptide

• Ribosome/mRNA complex dissociates

Termination and recyling in euks

• Similar to proksL euk release factors (eRFs) recognize stop codon + release polypeptide and tRNA

• eRF1 mimics a tRNA and binds the stop codon

• eRF3 through GTP hydrolysis helps release the polypeptide.

• ABCE1 uses ATP splits the subunits and recycling factors remove mRNA/tRNA so the 40S can be reused.

• eRF1 mimics a tRNA and binds to any of the 3 stop codons (UAA, UAG, UGA) via its N-terminal domain

• eRF3 is a GTPase that binds eRF1, GTP hydrolysis moves part of eRF1 into the PTC where it can promote polypeptide release

• eRF1 recruits ABCE1 which catalyzes subunit splitting via ATP hydrolysis = “power stroke” -

• Additional recycling factors remove the mRNA and tRNA from the 40S

• 40S is now able to be recycled for a new round of translation (recall circularization of mRNA thanks)

How is mRNA translation energetically expensive

• use as much as 80% of cells energy

• 10 aa> 300 kcal/mol

• 40 aa/sec by polyribosome ( multiplt ribosomes simultanosuely translating mRNA)

Polypeptide folding via molecular chaperones

• protein need to fold into correct 3D shapes before they can function

• Spon fold based on primary aa seq but cytoplasm BUSY place

• Hsp70→ new proteins

• Hsp60→ aka chaperonin→ unfolded proteins (heat shock)