Nucleic Acids Biochemistry Lecture 22 - Translation PDF

Recap of lecture • Alternative splicing increases number of proteins encoded by a genome • mRNAs can be spliced to exclude or extend introns in different combinations • Splicing allows 2 needed proteins to be made, amount varied by regulator • Steric hindrance excludes other splice site snRNP...

Recap of lecture • Alternative splicing increases number of proteins encoded by a genome • mRNAs can be spliced to exclude or extend introns in different combinations • Splicing allows 2 needed proteins to be made, amount varied by regulator • Steric hindrance excludes other splice site snRNP from a small intron • Invertebrates exploit alternative splicing to make antigen-recognition proteins • DSCAM exon#6 selection uses unique RNA:RNA hybrids and a repressor protein • Cell-specific mRNA splice variants made using genesplicing enhancers/silencers • Silencers use hnRNPs to prevent spliceosome binding to splice sites • Regardless of intron source, they now confer new capabilities for a genome • Exon shuffling patterns have been revealed by sequencing genomes • RNA editing alters mRNA sequence, protein by deaminating key nucleotides • Trypanosomal mitochondrial mRNA can be edited with U’s by guide RNAs • gRNAs uses 3 enzymes to insert U into the target mRNA to achieve RNA editing • Only processed mRNAs are exported to cytosol with aid of binding proteins • What does it take to make a protein? • How is a protein defined and started? • How does mRNA get made into protein? • What is the structure of a tRNA? • How do tRNAs do their job? • How do tRNAs maintain fidelity? • What is the ribosome? • How does it translate the bases to amino acids? • How are peptide bonds formed? BIOC 3041 Nucleic Acids Biochemistry Lecture 22 – Translation (mRNA, tRNA, tRNA + amino acids, & ribosomes) “Science moves on and so do its participants.” The discovers of aminoacyl-tRNA synthetase a) 1956, (left to right) Mahlon Hoagland, Paul Zamecnik, and Mary Stephenson. (b) Same characters, ~35 years later. Translation In rapidly growing bacteria protein synthesis accounts for: • 80% of cell’s energy and 50% of cell’s dry weight! Challenge of translation: • No specific affinity between amino acids and DNA bases The following bits of “machinery” are required for protein synthesis: • messenger (m)RNA (~5% of cellular RNA) • (aminoacylated) transfer (t)RNAs (~15% of cellular RNA) • aminoacyl tRNA synthetases • ribosomes (~100 proteins + 3-4 rRNA*s, *2/3 mass is RNA, rRNA≈80% of cellular RNA) • ancillary Protein "Factors" • a specially modified tRNA for Initiation Translation of 4-base RNA code into the code of 20 A.A.s requires adaptors Messenger RNA • Messenger RNA: Polypeptide chains are specified by open-reading frames • Open-reading frame (ORF) = protein coding region of mRNA Start codon • 3 in bacteria: AUG (sometimes GUG, or UUG) • AUG in eukaryotes… Stop codon • UAG, UGA and UAA ORF Frame? +1 Regular codon +2 +3 Start & Stop codons define protein-coding ORF in the mRNA Differences in mRNA structures in Pro-, Eu-karyotes Polycistronic mRNAs have multiple (poly) ORF, are usually bacterial • have a ribosome binding site (RBS) to recruit ribosome • RBS called Shine-Dalgarno sequence (3-9 bp on the 5′ side of start codon) • RBS is complementary to a sequence near the 3′ end of 16S rRNA RBS: ribosome binding site “Translational coupling” Monocistronic mRNAs contain only a single ORF, usually eukaryotic • Have Kozak sequence to recruit ribosome, increase translation efficiency 5′ cap recruits ribosome & then allows it to start “scanning” for start codon 3′ poly a promotes efficient recycling of ribosome mRNAs from bacteria, eukaryotes differ in RBS, ORFs per mRNA, modifications Transfer RNAs (tRNAs) • tRNAs are adaptors between mRNA codons & amino acids • Every tRNA has 5′-CCA-3′ at 3′ end where cognate amino acid is attached tRNA1 tRNA2 tRNA3 • Aminoacyl tRNA synthetase enzymes attach amino acids to tRNas • Up to 45 different tRNAs needed to bind all mRNA codons types • Unusual bases are present in tRNA structure tRNA modifications! • Ψ=pseudouridine (isomer of uridine) • dihydrouridine is reduced form, neither is essential to cell tRNAs have conserved 3′ termini that special enzymes append amino acids to tRNAs share common secondary structure resembling a cloverleaf All tRNAs have 1 stem, 3 stem-loops, and 4th loop; 1) The acceptor stem (accepts the amino acid) with CCA sticking out beyond paired bases (2) yU loop: 5’-TyUCG-3’ (3) D loop (rich in Dihydrouridines) (4) anticodon loop (pairs with mRNA codon) (5) variable loop: 3-21 bases D- & yU-loops interact, so do stems to stabilize tRNAs have functional and structural regions formed by RNA base pairing Attachment of amino acids to tRNA • tRNAs are ‘charged’ by adding an amino acid to tRNA’s 3’ terminal Adenosine nucleotide via a high-energy acyl linkage • Aminoacyl tRNA synthetase ‘charges’ tRNAs in two steps Step 1: adenylylation Activation of the Amino Acid aa + ATP aa-AMP + PPi Step 2: tRNA charging Aminoacyl group transfer to tRNA aa-AMP + tRNA aa-tRNA + AMP • Aminoacyl tRNA synthetase fall into 2 classes, differing by where amino acid attaches to tRNA and number of enzyme subunits tRNA charging done by sequential addition of ATP, then tRNA to amino acid tRNA synthetase recognizes unique structural features of cognate tRNAs • Each aminoacyl tRNA synthetase attaches single amino acid to tRNAs • “isoaccepting” tRNAs are synthetases that recognize, attach more than 1 type of tRNA to appropriate amino acid • Anticodon loop, acceptor stem allow tRNA synthetase to identify what type tRNA is and what amino acid to add • ‘Discriminator base’ encodes recognition information • Anticodon itself can’t be used for recognition since 1 amino acid encoded by multiple codons (e.g., Ser uses 6 codons, two are completely different!) Different tRNA structures confer a “2nd genetic code” to get the right A.A. added Aminoacyl-tRNA formation is very accurate • Some aminoacyl tRNA synthetases use an editing ‘pocket’ (as a molecular sieve) to charge tRNAs with high fidelity • A.As are small, similar so discrimination between some is difficult • Each amino acid fits into an active site pocket in the enzyme (for 1st charging step) • Amino acid forms network of hydrogen bonds, electrostatic, and hydrophobic interactions • Only amino acids with a sufficient number of favourable interactions bind.. • If incorrect amino acid-AMP is made by tRNA synthetase, it can be bound by editing pocket that will hydrolyze it (and not correct amino acid-AMP) tRNA synthetases use two recognition pockets to prevent mis-charging of tRNAs Aminoacyl-tRNA formation is very accurate • Size exclusion keeps non-cognate AAs that are too big out of the aminoacylation site • Pre-transfer, editing site can accept the activated AA, but rejected AAs are hydrolyzed to AMP and AA rejected Post-transfer, editing site can accept activated AA but rejected AAs are cleaved from the tRNA tRNA synthetases use two recognition pockets to prevent mis-charging of tRNAs Ribosomes can’t discriminate between correctly and incorrectly charged tRNAs • Emphasizes need for tRNA synthetase to use binding pockets to prevent mischarged tRNAs • Ribosome blindly adds amino acids even if they are on the wrong tRNA Genetic demonstration • Can mutate anticodon so it binds to the wrong codon for that tRNA, delivers the right amino acid to the wrong codon  misincorporation of an amino acid! Biochemical demonstration • Can test with cysteine-tRNA charged with Cys or Ala • tRNA will correctly bind with proper codon, but deliver wrong amino acid  misincorporation of an amino acid! Pre –ribosome quality control of amino acid-tRNAs needed to prevent errors The Ribosome • Responsible for making all cellular protein from mRNA information • Far more complex than RNA & DNA-making machinery due to difficulty • 50 different proteins (or more) and at least 3 RNAs form ribosome • Rate of DNA replication: 200-1000 nt/sec (fast due to genome size) • Rate of translation in prokaryotes: 2-20 amino acids/sec (paces RNA Pol transcription rate of 50-100 nt/sec ÷3 since both occur in same place ) • Rate of translation in eukaryotes: 2-4 A.A.s/sec (why? spatial separation…) Ribosomes are complex ribonucleoproteins that convert mRNA info to proteins Ribosome has large and small subunit Ribosome has 2 subunits 1) Peptidyl transferase center (makes peptide bonds) 2) Decoding center (charged tRNAs “decode” mRNA codon sequence) P D • Subunit names is from their sedimentation coefficients (S=Svedberg or sedimentation velocity that is determined by size & shape) • Prokaryotes have a 50S and 30S ribosome (together is 70Sdifference due to shape of whole ribosome when individual subunits associate, binding causes compaction) • Eukaryotes have 80S ribosome with 60S + 40S subunits • Ribosomal RNA (rRNA) has same nomenclature based on Svedberg units • Bacterial 50S subunit made up of many proteins and 5S, 23S RNA, 30S subunit made up of 16S RNA (remember, 16S RNA hybridizes to mRNA’s RBS site) • More numerous, but smaller proteins actually make up less of subunits since RNAs are so huge (nucleotides outweigh amino acids 3:1) Eukaryotic ribosomes are larger than prokaryotic, have ribonucleoprotein subunits Composition of the prokaryotic and eukaryotic ribosomes • Recognize that eukaryotic ribosomes are 80S (= 60S +40S subunits) • Recognize that prokaryotic ribosomes are 70S complexes (= 50S + 30S) • Appreciate this complexity, don’t need to know individual components Small Large Large & small subunits associate & dissociate during each cycle of translation • Ribosome cycle has binding of mRNA by decoder, association of large peptidyl transferase, elongation, dissassembly after stop codon reached • mRNAs with multiple ribosomes known as polyribosomes or polysomes • Allows more protein to be produced , can stack ribosomes 80 bases apart • Explains why there isn’t much mRNA in cell…  most of the RNA is rRNA that produces protein Ribosomes make 1 protein at a time, but can work simultaneously on a mRNA Peptide bond synthesis • New amino acids are attached to growing polypeptide chain at its C-terminal end (not vice versa) • Peptides bonds are formed by transfer of the growing polypeptide chain from one tRNA to another Two charged species of tRNAs : 1) Aminoacyl-tRNA: attached at its 3’ end to the carboxyl group of the amino acid peptidyl transferase reaction 2) Peptidyl-tRNA: attached to the carboxylterminus of the growing polypeptide Peptide bond is switched from outgoing to incoming tRNA to lengthen protein The ribosome has three binding sites for tRNA • rRNAs are both structural and catalytic determinants of the ribosome • Most ribosomal proteins are on ribosome’s periphery sites are at interface between 2 subunits • Ribosome’s functional core is mostly rRNA • 3 tRNA binding sites; E P A A site: to bind the aminoacylated-tRNA P-site: to bind the peptidyl-tRNA E-site: to bind the uncharged tRNA (E is for exit) • Channels through ribosome exist that allow the mRNA and growing polypeptide to enter and/or exit the ribosome Ribosome holoenzyme creates active sites and tunnels for making protein Channels through ribosome allow mRNA and growing polypeptide to enter and/or exit the ribosome A site: to bind the aminoacylated-tRNA P-site: to bind the peptidyl-tRNA E-site: to bind the uncharged tRNA (E is for exit) aa-tRNA  peptidyl-tRNA  empty tRNA • Note pronounced kink in mRNA between two codons at P and A sites • This kink opens the vacant A site codon for aminoacyl-tRNA interaction • Channel size only allows very limited folding of the newly made polypeptide • Channel size only allows unpaired mRNA to enter the ribosome too Ribosome provides kink in mRNA which facilitates tRNA entry, enzyme action Recap of lecture • • • • • • • • • • • • • • • Translation of 4-base RNA code into the code of 20 A.A.s requires adaptors Start & Stop codons define protein-coding ORF in the mRNA mRNAs from bacteria, eukaryotes differ in RBS, ORFs per mRNA, modifications tRNAs have conserved 3’ termini that special enzymes append amino acids to tRNAs have functional and structural regions formed by RNA base pairing tRNA charging done by sequential addition of ATP, then tRNA to amino acid Different tRNA structures confer a “2nd genetic code” to get the right A.A. added tRNA synthetases use two recognition pockets to prevent mis-charging of tRNAs Pre –ribosome quality control of amino acid-tRNAs needed to prevent errors Ribosomes are complex ribonucleoproteins that convert mRNA info to proteins Eukaryotic ribosomes larger than prokaryotic, have ribonucleoprotein subunits Ribosomes make 1 protein at a time, but can work simultaneously on a mRNA Peptide bond is switched from outgoing to incoming tRNA to lengthen protein Ribosome holoenzyme creates active sites and tunnels for making protein Ribosome provides kink in mRNA which facilitates tRNA entry, enzyme action www.youtube.com/watch?v=TfYf_rPWUdY Reading – MBPGF p 434 – p437, p439-449 , MBOG7 pg 509-528, MBOG pg 457-479 • Last Quiz on Wednesday • Covers Lecture 18 Regulatory RNAs (prokaryotic) up Lecture 22 Alternative RNA Splicing • Same format as before • Arrive early and start early (9:20 ish)