Lecture 10: Molecular Biology - Translation I (2021)

**[Lecture 10: Translation I -- Regulation of Protein Synthesis (Rachel Green)]** **[General ]** - **Speed:** 20 aa/sec in *E. coli*, 5-10 aa/sec in *S. cerevisiae* - **Fidelity:** between 10^-2^ and 10^-5^ - **Start and stop codons:** - AUG (Are U great?) - UAG, UAA, UGA (U are grea...

**[Lecture 10: Translation I -- Regulation of Protein Synthesis (Rachel Green)]** **[General ]** - **Speed:** 20 aa/sec in *E. coli*, 5-10 aa/sec in *S. cerevisiae* - **Fidelity:** between 10^-2^ and 10^-5^ - **Start and stop codons:** - AUG (Are U great?) - UAG, UAA, UGA (U are great, U are awesome, U get As) **[tRNA ]** **Structure** - D loop and T loop: help in 3D structure - Anticodon: interacts with codon for specificity of translation, does not recognize stop codon - Acceptor end: CCA interacts with elongation factor EF-Tu and ribosomal tRNA binding sites (large ribosome subunit) - Only about 30 tRNAs per cell, how does that code for the 61 sense codons possible in the genetic code? **Wobble pairing rules:** - 1^st^ and 2^nd^ codon positions match. - 3^rd^ position codon (= 1^st^ anticodon position \@3') can be: [U:G], and tRNA can have inosine, which looks like G, so I:C, I:U, and apparently I:A are okay. **[Aminoacyl-tRNA Synthetases]** - 20: one per amino acid - The "second genetic code"; accuracy of AA-tRNA addition is hugely important, if wrong amino acid is added, even if the DNA is accurate, the protein product could be mutated/non-functional - Editing site: remove wrong aa that's added to the tRNA - Aminoacylation site: take the aa using ATP, transfer to the corresponding tRNA - Two step loading: - Activation: the enzyme (aminoacyl synthetase) binds the amino acid -- AMP complex and is activated (uses ATP) - Transfer step: The enzyme then transfers this activated amino acid to the tRNA, AMP released - Reaction driven by pyrophosphate hydrolysis (PPi + water =\> 2 Pi) - 1. tRNA specificity: - 2. Amino acid specificity: - - **[Ribosome]**  - - - - - 1. 2. 3. - - - - - - **[Translation General]** - Initiation: IFs - Elongation: EFs - Termination: RFs (and Recycling) - A site = amino acid site, where incoming tRNA + next AA comes in - P site = peptide bond site (middle), where the peptide bond forms for next AA - E site = exit site, tRNA has donated its AA and exits ribosome, must be charged again with AA **[Initiation]** Find AUG and put initiator tRNA at P site, most divergent step - Prokaryotes: **Shine-Dalgarno** seq. = polypurine tract 6-8 bp upstream AUG that hybridizes to 16S rRNA (small subunit) via anti-SD, which is a polypyrimidine tract. Discovered with phylogenetics. - Three IFs are critical for bringing initiator tRNA - IF1 blocks the A site and IF3 the E site to direct the initiator tRNA to the P site. - GTPase of **IF2** involved in large subunit joining; when initiator tRNA finds AUG, GTP gets hydrolyzed - Initiator tRNAs in bacteria have the protection group f-Met (formyl-Met) that looks like a peptide and stabilizes it; the initiator tRNA doesn't bind any IFs. In eukaryotes, initiator tRNA binds initiation factors. - Eukaryotes: **Cap and polyA tail critical** - First: Protein complex (**eIF4E**, 4G, 4A, 4B) assembles at cap and **eIF4G** interacts with PABP =\> forms a closed loop for translation - Ternary complex: 40S subunit with eIFs; **eIF2** GTPase binds met-tRNA and GTP. - **eIF1** blocks the A site and **eIF1A** the E site - **eIF3** recruits ternary complex to **eIF4G** and scanning starts at cap complex - Deposition of initiator tRNA usually at first AUG encountered (\~90%) but depends on context, if fits [Kozak consensus] initiation site more likely - ***eIF2 hydrolyses GTP when start site is recognized*** - ***eIF5b hydrolyses GTP for large subunit joining*** (equivalent of IF2 in prokaryotes) **Note:** The Kozak sequence is NOT the functional equivalent in eukaryotes to the Shine-Dalgarno in prokaryotes. She likes to ask this, but it is NOT THE SAME AT ALL---there's no complementarity in the small subunit, it just provides a "favorable context". **[Elongation]** The cycle required to extend the growing peptide chain by one amino acid - 1. **EF-Tu**/e*EF-1a* (GTPase) binds tRNAs to load into ribosome. EF-Tu is probably most abundant protein in biology! - - - - - - 2. Peptide bond formation = classic ribozyme; no proteins involved - 2'OH or 3'OH esterification of amino acid to tRNA is labile - Nucleophile on amino-acyl tRNA at A attacks labile aminoacyl ester bond at P - Growing peptide transferred to A. - 2'OH of P site is most important for positioning (not active site nucleotides of ribosome) - Targeted by antibiotics, resistance arises by mutation of rRNA gene, r-proteins and acquisition of rRNA methylase 3. Translocation (**EF-G**/ *eEF-2*) (GTPase) - Movement of mRNA:tRNA complex on the small subunit by 3 nucleotides, from the A to the P site. - EF-G hydrolyzes GTP and promotes translocation. **[Termination]** Recognizing the termination codon and releasing the polypeptide chain. tRNAs don't recognize stop codons. ***Termination in bacteria and eukaryotes has evolved independently.*** - Prokaryotes - RF1 recognizes UAG/UAA STOP codons and RF2 recognizes UGA/UAA STOP codons. These release factors somehow stimulate hydrolysis of the polypeptide chain. - RF3: GTPase that stimulates RF1 and RF2 activity - Eukaryotes - eRF3 and eRF1 form a complex. - eRF1 recognizes all stop codons. - eRF3 hydrolyses GTP, releasing the polypeptide chain. **[Ribosome recycling]** Need to split the small and large subunits to be able to start a new round of translation. - Prokaryotes - RRF, EFG, IF3 - Eukaryotes - eRF1/3, ABCE1 **[Polysomes]** - Translation occurs on polysomal mRNAs  - With polysome profiling, RNA absorbance on Y axis and fraction (after centrifugation in sucrose gradient) on X axis - 30S and 50S subunits, unbound to mRNA, are less massive and so appear higher up in the gradient after centrifugation - Allows for study of translation dynamics -- how many ribosomes are translating an mRNA? - Polysome profiling different from ribosome profiling: in ribosome profiling, mRNA in the cell are subjected to treatment with a nuclease. mRNA protected by ribosomes (actively being translated) remain and can be sequenced. **[Nonsense Mediated Decay -- premature stop codons]** **What:** NMD may occur at the [pioneer round of translation] - Pol II transcripts acquire CBP20/80 (nuclear cap binding proteins) eventually replaced by eIF4E in the cytoplasm, and PABP2 (nuclear poly(A) binding protein) eventually replaced by PABP1. - These can be defined as components of the translation initiation complex in the early rounds---possibly the first (pioneer) round of translation in mammalian cells. - Rationale of a pioneer round of translation during which NMD functions 🡪 NMD surveys mRNAs to eliminate those that prematurely terminate translation. The earlier the better to avoid synthesis of truncated proteins. **How:** - IP assays show CBP80, not eIF4E, associates with exon junction complex (EJC) components and the up frameshifts (UPFs) - Debated, since more recently, it has been shown that NMD works both with CBP80 and eIF4E bound to mRNPs **[Current Model for NMD ]** ***It is the EJC that provides position information needed to discriminate premature stop codons from natural stop codons. Recognition of premature termination codons (PTCs) appears to be dependent on the definitions of the exon-exon junctions.*** 1. Un-spliced transcript in the nucleus with PTC. 2. Spliced transcript carries exon junction complexes (EJCs) at each exon-exon junction, marking the sites that have undergone splicing 3. Ribosomes translating the transcript displace EJCs until a stop codon is encountered. Upon stop codon recognition by eRF1-eRF3, interactions between the termination complex, UPFs and the EJC occur. 4. This in turn recruits factors responsible for the degradation of the faulty mRNA transcript. **[Targeting NMD in Genetic Diseases ]** **Problem:** Mutations leading to PTC in cystic fibrosis, muscular dystrophy 🡪 give rise to truncated protein **Solution:** suppress mutation with drug allowing for read-through of PTC and therefore expression of full-length protein. - Aminoglycosides (used to kill bacteria) - PTC124 in clinical trials for CF, MD, hemophilia **Side-effects:** very toxic systemically \*\*much more on mRNA processing and splicing in a later lecture\*\*