BIOL2010 Translation Lecture 1 2024 PDF

anslation – Lecture 1 Module: BIOL2010 The Flow of Genetic Information Kif Liakath-Ali PhD www.splicelab.co.uk Central dogma of molecular biology DNA makes RNA makes Protein Protein sorting  Translation...

anslation – Lecture 1 Module: BIOL2010 The Flow of Genetic Information Kif Liakath-Ali PhD www.splicelab.co.uk Central dogma of molecular biology DNA makes RNA makes Protein Protein sorting  Translation Lectures Lecture 1 – Today Lecture 2 – 13/12/2024 (06/12/2024) - Ribosomes - Introduction - Translational control - Steps - 10 mins Break - Building blocks: tRNA and charging Lecture 3 – 10/01/2025 - Review of Translation (KLA) - Overview of exam (DAD)  Learning outcome 1. Overview of Translation Explain the purpose of the translation process in protein synthesis within the central dogma of molecular biology. Identify the stages of translation 2. Molecular Components Describe the roles of ribosomes, mRNA, tRNA, and aminoacyl-tRNA synthetases in translation. Understand the function of codons in mRNA and their recognition by anticodons on tRNA. 3. Genetic Code Define the characteristics of the genetic code (e.g., redundancy, universality, start/stop codons).  Translation - Introduction Translation or Protein synthesis: The mechanism by which RNA (produced by transcription) is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein Protein production = Protein synthesis, mRNA translation Protein translation  Translation - Introduction Key Concepts Translation requires cooperation between ribosomal RNAs, transfer RNAs, messenger RNAs, and numerous proteins. Translation is performed by one of the largest molecular complexes in cells, the ribosome. The steps of translation are grouped into three stages: initiation, elongation, and termination. These are very different from the identically named stages of transcription.  Translation - Introduction Transcription vs. Translation Easy way to remember! The word “Transcription” appears before “Translation”. In a cell (for a given gene), transcription happens before translation  Translation - Introduction Major points  Protein synthesis is similar in all organisms  We’ll compare prokaryotic and eukaryotic differences  Catalysed by a ribozyme (ribosome + RNA)  We’ll look at the how amino acids are joined together  Has to be very accurate (1 error/10,000 amino acids)  We’ll see which of the many steps/components is the most important in ensuring high fidelity  It is important to control protein synthesis tightly  We’ll look at the consequences when it goes wrong  After synthesis, proteins need to be folded, modified and/or targeted correctly  See other lectures in BIOL2012 and BIOL2056  What do we need for protein synthesis?  A template that has sequence (clue/code) on where to start decoding  A supply of building blocks  Something to assemble the building blocks into chains  A way to supply the correct building block at the appropriate time  Rules to define HOW to decode (including where to start and stop)  Energy to drive the process  What do we need for protein synthesis? Ribosomes are the synthetic machinery (rRNA and proteins) The template is a mature mRNA (capped and tailed in eukaryotes) All 20 amino acids are activated as aminoacyl-tRNAs Requires a large number of other enzymes/proteins (factors) and energy in the form of GTP and ATP Adding each amino acid ‘costs’ 4x ATP Translation – At a glance  Key players in translation: Ribosome, tRNAs, mRNA and Translation factors Ribosome 1 Angstrom (Å ) = one ten-millionth of a millimeter The relative sizes of components of the cellular translation machinery  General ribosome structure  Small and large subunits must join, otherwise no translation  Association creates three sites for tRNA to occupy  mRNA slides through a channel which is on small subunit Dark red = 23S rRNA Orange red = 5S rRNA Pink = proteins of large subunit Dark blue = 16S rRNA Light blue = proteins of small subunit A: Aminoacyl-tRNA P: Peptidyl-tRNA E: Exit Ribosome – One of the largest macromolecules https://learn.genetics.utah.edu/content/cells/scale/  mRNA - the template for translation  Composed of ribonucleotides (A, C, G and U)  Contain non-coding or untranslated regions (NCRs, UTRs) at their 5’- and 3’-ends  Template needs to define exactly where to start (determines ORF)  The genetic code uses three RNA bases to specify one amino acid (triplet code)  Each of the 20 amino acids is specified by one or more triplets  The protein is synthesised from a continuous sequence of non-overlapping CODONS running from an initiation codon to a termination codon T AR OP ST 3’ ST 5’ 5’-UTR 3’-UTR AAAAAAAAAAA mG7 coding region (cap; eukaryotes) (poly(A) tail; eukaryotes) ‘open reading frame’ [ORF] regulatio regulatio n n  Types of RNAs to remember Messenge Transfe Ribosom r r al  Translation Occurs in Three Stages Initiationrequires base pairing between mRNA and tRNA Elongationof polypeptide occurs when an amino acid is added to the carboxy-terminus of the polypeptide in the P site Terminationoccurs when the bond holding the polypeptide to tRNA is hydrolyzed Hydrolysis - the chemical breakdown of a compound due to reaction with water  Stage 1: Initiation requires base pairing between mRNA and rRNA Goal = bring all of the elements necessary for translation together into a giant cluster Ribosomal subunit to find ribosomal binding site = initiation site = Kozak sequence Once the mRNA and small subunit are properly aligned, the first tRNA (initiator tRNA) binds to the AUG, and the large ribosomal subunit clamps down on the small subunit, forming an intact ribosome  Stage 1: Initiation requires base pairing between mRNA and rRNA - Each stage of translation requires other protein factors - Initiation Factors (IFs/eIFs) - positioning of the small ribosomal subunit (and first aminoacyl-tRNA) at the initiation codon - joining of large ribosomal subunit to make whole ribosome - Initiation is the slowest step therefore limits rate of translation and is the step where most translational control occurs Large (60S) ribosomal sub-unit mRNA AAAA AUG UAG UAA UGA Small (40S) ribosomal subunit  Stage 2: Elongation cycle during translation Amino acid is added to the carboxy terminus of the polypeptide in the A site The elongation cycle  Stage 2: Elongation cycle during translation Elongation Factors (EFs/eEFs) - Ensures correct amino acid is added sequentially to the growing protein chain by base pairing of tRNA with mRNA - Decodes 10 – 40 aa per sec with only 1 in 10,000 error rate - Bring in the next tRNA - Join the second amino acid onto the first - Move along to the next triplet - Expel the first tRNA - Keep doing these cycles until… mRNA AAAA AUG UAG UAA UGA  Stage 3: Termination Termination occurs when the bond holding the polypeptide to tRNA is hydrolyzed Stop codon  Stage 3: Termination Termination Factors (RFs/eRFs) - release of completed polypeptide when the stop codon is reached - No tRNA corresponding to the stop/termination codon - Bring in a factor to release the completed polypeptide Recycling (RRF in prokaryotes, ABCE1 in eukaryotes) - ribosomal subunits detach and are kept separated to allow new round of translation mRNA Completed polypeptide AAAA AUG UAG UAA UGA 60S 40S Dissociated subunits Initiation – role of the 5’- cap eIF3 equivalent to IF3, but 13 subunits Binds eIF4G and the 40S subunit PABP = poly(A)-binding protein  Synergism between 5’ and 3’ ends Open Reading mRNA 5’ UTR Frame (ORF) 3’ UTR AAAA m7G cap Poly(A) tail  mRNA transcribed in vitro, in combinations with or without m7G cap or poly(A) tail  Added to rabbit reticulocyte lysate (contains ribosomes and other factors required for translation) in presence of radioactive methionine  Products of in vitro translation separated by SDS- PAGE and visualised with autoradiography  Initiation in eukaryotes  Eukaryotic initiation differs considerably from bacterial initiation  eIF2 + the small 40S ribosome subunit + methionyl-tRNAimet + GTP bind to each other  The 40S subunit binds to mRNA with other initiation factors (eIFs) bound to the cap and poly A tail regions  The 40S ribosome then “scans” the mRNA looking for the AUG initiation codon: usually uses first AUG it encounters  More efficient if this AUG is within the Kozak Consensus sequence  eIFs then dissociate and the 60S subunit binds  Intact cap and tail regions are essential for initiation  Protein synthesis in eukaryotes - Initiation 48S pre-initiation complex (PIC) formation and mRNA circularisation Binds poly(A) tail PABP Scaffold for AA AAA complex AAA Provides first tRNA Met assembly 4B 2 GTP 1 1A eIF4G 4E 4B mRNA 4A 3 40S AUG m7G cap-binding Mnk Similarities to Unwinds RNA Bridges scaffold to prokaryotes: structures  eIF2 bound to GTP and ribosome brings in first tRNA Key differences:  eIF3 and eIF1 ensure  mRNA is circularised accuracy of initiation and  Complexity adds several opportunities for prevent association of 60S control large subunit  Control of eIF4E availability Rapamycin mTORC1 Primed PP P P P P 4E-BP1 4E-BP1 4E-BP1 4E 4E 4E 4E-BPs contain a similar sequence motif 4E to eIF4E binding site of eIF4G Hyperphosphorylation of 4E-BP disrupts interaction with eIF4E eIF4G Key control point:  Without the eIF4E:eIF4G interaction, how can the ribosome attach to the 28  Control of eIF2 availability  48S pre-initiation complex (PIC) formation and mRNA circularisation PABP AA AAA AAA Provides first tRNA Met 4B 2 GTP Coupled to GTP 1 1A eIF4G 4E 4B mRNA 4A 3 40S AUG Mnk Key control point:  Without the first tRNA, how do you start synthesising the polypeptide?  Function and control of eIF2 eIF2 has three Met g subunits Ternary complex  Met- gamma binds b GTP tRNAi Met 2 GTP eIF2 GT P GDP finds and guanine binds 40S recognise nucleotide eIF2B subunit s exchange Pi AUG start GTP codon eIF2 GD Translation P Initiation Release of factors GTP hydrolysis  Function and control of eIF2 eIF2 has three Met g subunits Ternary complex P  gamma binds Met- b tRNAi Met GTP phosphorylated 2 GTP eIF2 GT on Ser51 of P alpha subunit GDP eIF2B Pi GTP eIF2 GD Translation P Initiation PERK, PKR, Release of GCN2, HRI factors P  Multiple eIF2 kinases 2xRBD kinase domain PKR RBD = RNA-binding domain Mammals HRI Haem-binding Mammals HisRS (aa-tRNA synthetase) GCN2 Mammals, yeast, flies PERK Mammals, flies BiP-binding TM kinase ‘Paradoxical’ increase eIF2α eIF2 α eIF2B activity & in translation of Stress kinase phosn. translation initiation certain specific mRNAs General translation  Activation of PKR in response to viral RNA Virus-infected cells produce Interferon (IFN) This induces PKR (protein kinase R) expression in neighbouring cells (‘anti- IFN viral state’) dsRNA Inhibitory domain Based on Hinnebusch (2005) Nature Structural & Molecular Biology 12, 835 - 838  This causes a reduction in protein synthesis of both host and viral mRNA, which should prevent further infection  Protein synthesis in bacteria - Initiation Only AA-tRNA to enter ribosome at P [peptidyl]-site  mRNA binds a special formylmethionine-tRNAf + the 30S subunit at the P-site using initiation factors IF1, IF2 and IF3 and GTP.  IF1 – binds in A site, prevents elongator tRNAs entering  IF2 – binds the GTP and the fMet-tRNAf  IF3 – prevents association with 50S, helps ensure fidelity of initiation codon selection (not present in all bacteria)  This gives the “initiation complex”  Sequences in bacterial mRNA help the ribosome locate the initiation codon  Prokaryotic mRNAs possess a Shine-Dalgarno sequence that base pairs to the 3’- end of the 16S rRNA  This places the start codon AUG at the ribosome P-site about 10 bases 3’ of the S-D sequence Helps ensure accuracy: permits use of polycistronic mRNAs  This is followed by the binding of the 50S subunit and dissociation of IF1 and IF3.  Protein synthesis in bacteria - Initiation  IF1 and IF3 dissociate once the 30S is at the initiation codon  This is accompanied by the binding of the 50S subunit and hydrolysis of the GTP bound to IF2, causing IF2 to also dissociate  This gives the 70S ribosome for elongation with the first tRNA in the P site, and an empty A site ready for the next tRNA  Differences in eukaryotic messenger RNA Eukaryotic mRNAs contain several important features:  - a cap (made from a modified G nucleotide) and a poly(A) tail  - No Shine-Dalgarno sequence  - 5’ and 3’ UTRs may contain several features (sequence or structure- based)  that help regulate protein expression/mRNA stability/localisation Open Reading Frame (ORF) mRNA 5’ UTR 3’ UTR AAAA AUG UAG m7G cap UAA Poly(A) tail Initiation UGA codon Termination codon  Summary  Translation is the synthesis of chains of amino acids into protein by decoding the message that was stored in the DNA (therefore change of “language”)  mRNA is the intermediate, produced by transcription (nucleotide language remains similar)  Initiation is the positioning of the small ribosomal subunit at the initiation codon, and the joining of the large ribosomal subunit  Prokaryotic initiation uses rRNA:mRNA base pairing to define the start point, eukaryotic uses a sequence consensus  Eukaryotic initiation is more complex but more tightly controlled  Summar y  Eukaryotic translation initiation is controlled by multiple mechanisms  Availability of cap-binding protein  Availability of ternary complex (contains first Met-tRNAi)  Proteins are sometimes also transported during translation  Accumulation of misfolded proteins causes activation of PERK  Next lecture – how amino acids are added to the correct tRNA 10 mins break How amino acids are added to the correct tRNA? Amino acids - Building blocks of proteins Central (α) H carbon Amine O Carboxylic H2N Cα C Acid OH R Variable R group  The 20 proteinogenic amino acids  Polar = Hydrophilic  Acidic – COO- provides negative charge H  Basic – N+ provides positive charge O  Uncharged polar – OH provides polarity H 2N Cα C  Nonpolar = Hydrophobic OH  R group normally contains just C and H R  The 20 amino acids give properties that are essential for the formation of proteins Small Tiny Hydrophilic P (polar) Hydrophobic (non-polar) G A N S C -ve charge V I T D L E M Aliphatic K Q F Sulphur- H R W containing Y +ve charge Aromatic  The Genetic Code  The genetic code is universal* with some minor variations (e.g. mitochondria) – indicating life arose from one original ancestor  *Organisms exhibit codon usage bias (a preference to decode certain codons and not others) – important to consider if making e.g. human protein in E. coli mRNA AAAA AUG UAG UAA UGA  The Genetic Code  The genetic code is triplet, continuous and non-overlapping  If one RNA base specified one amino acid, only 4 amino acids could be encoded – one amino acid for each base.  If a pair of any of the four RNA bases specified one amino acid, then only 16 amino acids (42) could be encoded.  A triplet of any of the four bases can specify 64 combinations (43), therefore provides more than sufficient triplets to specify each of the 20 amino acids.  Addition or deletion of single nucleotide bases changes the frame downstream therefore the code must be continuous  The continued contact of tRNAs with the mRNA template during elongation steps means that the code cannot be overlapping  Second Base Third Base First Base  e.g. What is the consequence of a cytosine deamination mutation in each of the three bases? (cytosine-uracil)  D614G missense mutation in SARS-CoV-2  D614G caused by a single base change  Initially rare, but increased after March  Improved infectivity  Replicates more readily in nasal passages and trachea but not lungs Plante et al., Nature 2020  Mink “cluster 5” has deletion of two amino acids (69-70), Y453F, I692V, M1229I  The adapter molecule – tRNA  Most tRNAs contain between 70 and 90 bases  They all have highly conserved tertiary structures (L-shape) with much internal base pairing  The 3’-terminus of every tRNA ends in CCA, with each amino acid linked as an ester to the 3’-OH (or in some cases to the 2’-OH) of the last A  tRNAs contain numerous unusual post-transcriptionally modified bases  tRNAs possess an ANTICODON that recognises the mRNA CODON by anti-parallel base pairing Cracking the genetic code  tRNA Secondary and Tertiary Structures H- bonds 1 = acceptor stem 2 = D loop 3 = anticodon loop 4 = TψC loop (thymidine, pseudouridine, cytidine) tRNA secondary structure (e.g. – yeast tRNAPhe) Base methylation  Modified bases in tRNAPhe Pseudo-uridine  Pseudouridine (ψ) has more possibilities for H-bonding (a,d), and more rigidity  Better for stability in tertiary structure and interaction with ribosome H-bonds  Amino acid activation and formation of aminoacyl-tRNA esters  The peptide bond -CO-NH- linking amino acids is an amide bond and its synthesis requires the input of energy  Each of the 20 amino acids is first linked to the 3’-OH (or 2’-OH) of the terminal adenosine of a specific tRNA by an ester bond (gives an amino acyl-tRNA)  The hydrolysis of the ester bond helps drive peptide bond formation in the ribosome  To ensure an amino acid is only linked to its correct tRNA, highly specific enzymes are used. These vital enzymes are called amino acyl-tRNA synthetases  These enzymes ensure that a tRNA is linked to its correct amino acid so must show high specificity and fidelity – How?  Amino acyl-tRNA synthetases  tRNA synthetases recognise the tRNA and the amino acid  They catalyse the reaction of the terminal adenine ribose (of CCA) with the amino acid carboxyl group to give NH2CHRCO-3’O-tRNA  Two classes of synthetases exist (I and II)  Activation of an amino acid occurs in a two-step process 1. Amino acid + ATP + enzyme enzyme-AMP-amino acid + PPi high energy bond ‘ACTIVATED INTERMEDIATE’ 2. enzyme-AMP-amino acid + tRNA Aminoacyl-tRNA + AMP + enzyme  Each time, the equivalent of two ATPs is used (the enzyme uses a second to drive the reaction)  Overall error rate is only 1 in 10,000  TWO-step reaction allows PROOFREADING – mistakes made in Reaction 1 are eliminated in Reaction 2  Proofreading by amino acyl-tRNA synthetases e.g. by tRSVal larger aa, cannot enter acylation site, e.g. Phe Phe not attached to tRNA (catalyti (editing tRNAVal c domain) domain) acylation hydrolytic active site active site tRNAVal Ala Ala tRSVal acylation hydrolytic active site active site smaller aa, e.g. Ala enters acylation site, forms tRSVal misacylated tRNA, which Phe CAN enter second site so tRNAVal hydrolysed Val Ala tRNAVal correct amino acid, Val Val enters acylation site, acylation hydrolytic forms active site active site properly acylated tRNA Val which cannot enter tRSVal hydrolytic site = correctly charged Val-tRNAVal  The anticodon plays no role in loading of correct amino acid onto the tRNA but is essential for correct translation of code  Therefore if an amino acid is linked to the wrong tRNA, then the amino acyl- tRNA still recognises the anticodon and inserts the wrong amino acid. amino acid attached here recognises the codon in the mRNA  Experimental proof that the anticodon recognises the mRNA codon not the amino acid  Cysteine-tRNACys is prepared  Raney nickel (mix of Ni and Al) reduces cysteine’s -CH2-SH group into -CH3 CH2 CH3 (alanine) SH  Therefore cysteine-tRNACys is converted into Cys, C Ala, A alanine-tRNA Cys  The modified alanine-tRNACys is used in the synthesis of haemoglobin in an in vitro experiment  The result is that alanine is incorporated into the haemoglobin in place of each cysteine (UGU or UGC codons)  Thus the anticodon is recognised, not the amino acid (alanine)  Therefore accuracy/proofreading in aminoacylation of tRNAs is critically important  “Wobbl e”  A term coined by Crick that defines the ability of the first base of an anticodon to base pair with more than one codon  Allows an aminoacyl-tRNA to dock with more than one codon, so that fewer than 61 tRNAs are needed to decode the 61 codons  Wobble generally involves the nucleotides U or inosine (I) at the first position of the anticodon (matches 3rd position of codon)  Unusual Base-pairing in RNA  G pairs with C  A pairs with U  U can also pair with G U:G  Therefore: - U in the first anticodon position 3’ 5’ 3’ 5’ can base pair with A or G in the 3rd position of the codon tRNA - G in the first anticodon position can base pair with C or U Hence, synonymous pairs mRNA 5’ 3’ 5’ 3’ of codons end U/C or A/G (Look at pairings in the codon table)  e.g. tRNA Phe has 5’ GAA 3’ in anticodon can decode UUU and UUC Second Base Third Base First Base  Further degeneracy of base-pairing in first position of anticodon  Some anticodons of tRNA have inosine (I) in the 1st position (5’-position).  Inosine (I) looks like guanosine without an amino group but is made by deamination of adenine  This conversion is done AFTER transcription of the tRNA guanosine tRNA  Extra base pairs involving inosine  I can base pair with A, U or C using 2 H- bonds A:I C:I U:I  Examples of wobble binding  If the first base of the anticodon is inosine (I) then it can base pair with A,U or C.  Therefore a single tRNA can recognise MORE than one codon  e.g. the 5’-IGG-3’ anticodon of tRNAPro can recognise three of the proline codons: 3’ 5’ tRNA 3’ GGI GGI GGI 5’ tRNA 5’ CCU CCC CCA 3’ mRNA Ile AUU A single tRNA can also decode the Ile AUC three Ile codons Ile AUA mRNA 5’ 3’ Met AUG By exploiting ‘wobble’, bacteria can decode 61 codons using just 31 tRNAs In mitochondria, even fewer tRNAs are required (22 in humans)  Summary of key roles of tRNA  To activate the amino acid as a tRNA ester  To act with the correct synthetase to ensure that the correct amino acid is linked to its tRNA  To base pair its anticodon triplet with the correct codon of mRNA - decoding role (anticodon:codon pairing can “wobble”)  To bring the correct amino acid to the ribosome  To facilitate peptide bond (-CO-NH-) formation of the nascent chain and the next amino acid  Further Resource Protein synthesis https://www.ibiology.org/biochemistry/protein-synthesis/  Next lecture… Ribosomes, the cellular factories that manufacture proteins