Lecture 17: Translation I

Translation I Side notes: Translation: synthesizing proteins from mRNA Translation is the process by which a polypeptide chain is synthesized from an mRNA template and is the final step in the flow of genetic information Proteins are made from 20 different amino acids at a rate of 15 amino acids per second (error rate 10^-3 to 10^-4) Ribosomes are the main macromolecular machines that carry out translation Transfer RNAs (tRNAs) link mRNA to its amino acid sequence Translation has four main stages: initiation, elongation, termination, and ribosome recycling Transfer RNAs (tRNAs) link codons to protein sequence through their structure tRNAs are small (75-94 nucleotides) and specific to amino acids tRNAs have four base-paired regions called stems and three singlestranded sequences called loops The 5’ and 3’ ends form the acceptor stem that is followed by a conserved 3’ CCA tail, which becomes the attachment point for amino acids The anticodon loop has 3 nucleotides called anticodon that base-pair with the codon on mRNA Because of this base-pairing, tRNA physically links amino acids to their respective triplet codon tRNA is depicted as a clover-leaf, but it’s 3D structure of tRNA takes on an L shape, which contributes to interactions, like base-triple interactions tRNA has modified nucleotides - for example, the DHU (D) loop is named after dihydrouridine (D) The TpsiC (T) loop has ribothymidine (T) and pseudouridine (psi) A hyper modified purine occurs just after the anticodon, to prevent this from base-pairing with the codon in mRNA. This helps align the codon and anticodon properly Inosine (often found in anticodon loop) is important for wobble pairing The genetic code: amino acids are encoded by triplet nucleotide sequences A triplet nucleotide sequence is called a codon Some amino acids are specified by a single codon, some are specified by multiple codons Some codons do not code for amino acids, but halt translation and are called stop codons RNA doesn’t recognize all the codons for a specific amino acid. Different tRNAs for the same amino acid are called isoacceptors Codons are mostly conserved, but there are exceptions, such as start codon in mitochondria (AUG and AUA) or in mycolasmatryptophan (UAG) A single tRNA can decode several different codons due to wobble pairing 1st and 2nd positions of mRNA (codon) follow strict Watson-Crick pairing via antiparallel mini-RNA helix 3rd position of codon allows promiscuity called wobble pairing where nonWatson-Crick pairing is permissible. If inosine is present in anticodon, it can pair with U, C, or A Wobble pairing means the same tRNA can interpret both UUU and UUC for example Because of these interactions, 40 tRNAs are typically found in an organism and decode 61 codons Translation I Side notes: How are tRNAs linked to their amino acid? • A specific aminoacyltRNA synthetase catalyzes the addition of an amino acid based on the features of tRNA • Any aminoacyl-tRNA synthetase catalyzes the addition of any amino acid to any tRNA • Any amino acid will bind to tRNA and a specific aminoacyltRNA synthetase Aminoacyl-tRNA synthetases are key enzymes needed for coupling specific amino acids to the right tRNA Aminoacyl-tRNA synthetases (notation is aaRS, GlyRS or glycol-tRNAsynthetases) attach amino acid o tRNA in a two-step process requiring ATP hydrolysis Amino acid is activated by attachment of AMP Enzyme then transfers activated amino acid to tRNA 3’ CCA tail The tRNA that becomes loaded with an amino acid is denoted as tRNA(Met), tRNA(Gly), etc The aminoacyl-tRNA synthetase detects the tRNA based on specific sequence and structural features referred to as identity elements, found near the anticodon loop and acceptor stems Aminoacyl-tRNA synthetases select their cognate amino acid base on chemical features (charge, hydrophobicity, size, shape) using its active aminoacylation site Aminoacyl-tRNA synthetases have an additional site called the editing site which helps improve fidelity by rejecting the amino acid pre- or posttransfer Ribosomes are multi-component molecular machines that synthesize proteins Ribosomes are primarily composed of two-thirds ribosomal RNA (rRNA) and one-third ribosomal proteins (r-proteins) Ribosomes have two subunits: a small subunit and a large subunit The small subunit mediates interactions between mRNA and tRNA The large subunit catalyzes bond formation and has an exit tunnel for the growing polypeptide chain Ribosome structure and function are both relatively well conserved but differ in composition Localization of rRNA and r-proteins in ribosomal subunits Interface of small and large subunits is mainly rRNA Solvent face is enriched in protein, which extend “arms” into the RNA regions Highly basic “arms” of proteins help pack negatively charged rRNA phosphate backbone Ribosome composition throughout phylogeny Bacterial ribosomes represent the “core” ribosome structure With increasing organism complexity, more layers or expansion segments are added to ribosomes Archaea have an additional layer of proteins Lower eukaryotes have a layer of both protein-RNA Higher eukaryotes have an extra layer of RNA rRNA is organized into domains in large and small ribosomal subunits The rRNAs in the ribosomal subunits are divided into domains: domains of large subunit (23S and 5S rRNA) are more interwoven than those of small subunit (16S rRNA) Ribosomal RNAs are critical for ribosome function The decoding region of 16S rRNA mediates the interaction between tRNA and mRNA The 23S rRNA in the peptidyl transferase center interacts with the tRNAs Translation I Side Notes: Translation process Translation involves four main steps: Initiation Elongation Termination Ribosome recycling Each step is facilitated by protein factors for accuracy and efficiency! Protein factors facilitate each step in the translation cycle by two general mechanisms Many factors are GTPases (G proteins) that catalyze the hydrolysis of GTP GTP hydrolysis provides energy Their resulting conformational changes are linked with progression of ribosome through transition cycle Example: EFTu depends on GTP hydrolysis during elongation Other translation factors mediate interactions through their binding to ribosome Example: initiation factors IF1 and IF3 bind to A and E sites, which helps bind tRNA to the P site and prevents large and small subunits from associating too early Example: some even mimic tRNA to gain access to their interior: EFG binds in the A site and is similar to EFTu-tRNA Translation factor GTPases undergo conformational changes upon GTP hydrolysis GTPases are related and a P-loop motif (Gly-X-X-X-X-Gly-Lys) This motif interacts with the phosphates on GTP, and undergoes conformational changes as GTP is hydrolyzed Identity of bound nucleotide (i.e. GTP or GDP) dictates the conformation of the GTPase, and thus the ligand it interacts with EFG-GTP recognizes pre-translocation state ribosomes, and the EFG-GDP form dissociates from the ribosome Rate of GTP hydrolysis or exchange can be slow, GTPase-activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs) can promote these steps Ribosomes have three binding sites for tRNA: aminoacyl, peptidyl, and exit sites Each aminoacyl-tRNA first binds to the aminoacyl (A) site, followed by the peptidyl (P) site, and finally the exit (E) site Initiation: AUG start codon and initiation factors Three steps of initiation The small ribosomal subunit identifies the start codon in a mRNA The AUG marks the star of an open reading frame and is recognized by initiation factors (IFs) and a special initiator methionine tRNA A methionyl-tRNA loads onto the P site of the ribosome, base-pairing with the start codon The large ribosomal subunit joins the complex The ribosome is now ready to move along the mRNA Translation initiation: shared features of initiation tRNAs in bacteria and Translation I Side notes: Which factor(s) directly bring tRNA(fMET) TO P site in bacteria? • • • • • • IF1 IF2 IF3 IF1 and IF3 All of the above None of the above eukaryotes Both have the same initiator codon (AUG), which is decoded by the initiator tRNA (preventing association with EFTu/eEF1A) Both initiator tRNAs have identity elements: three G-C base pairs in anticodon stem The initiator tRNAs are loaded onto P site Identifying the start codon in bacteria: Shine Dalgarno Sequence Bacterial mRNAs are often polycistronic - having several open reading frames, each with its own start and stop codon Initiation codons are preceded by a Shine-Dalgarno sequence (aka ribosome-binding site) - this is a polypurine tract 6-8 (consensus sequence AGGAGGU) bases upstream of the initiator AUG Shine-Dalgarno pairs with a polypyrimidine region in the 3’ end of the bacterial 16S rRNA (the anti Shine-Dalgarno sequence), guiding the initiator AUG into the ribosomal P site Sequence deviations result in weaker interactions with the ribosome, influencing how efficiently mRNA is translated Bacterial translation initiation IF1 and IF3 independently associate in A and E sites, leaving P site unfilled tRNA(fMET) associates with mRNA and becomes directed to the P site IF2 is a GTPase, which hydrolyzes GTP to provide energy for joining large and small ribosomal subunits When subunits combine, IF1, IF2, AND IF3 are all displaced, marking the completion of initiation Eukaryotic translation initiation is significantly different than that of bacteria Eukaryotic mRNAs are not polycistronic like bacterial mRNA, but are monocistronic - only usually encode one protein There are no eukaryotic equivalents of the Shine-Dalgarno and anti-ShineDalgarno sequences The small 40S ribosomal subunit does not directly bind to mRNA upstream of AUG Many more initiation factors are needed (>11 distinct factors: elF1, 1A, 2, 2B, 3, 4A, 4B, 4E, 4G, 5, 5B) Initiation usually (~95% of time) occurs at the first AUG downstream of the 5’ end of mRNA and is found via ribosome “scanning” However, the context of the AUG influences how efficiently it will be recognized by the scanning ribosome The preferred sequence of the start codon is the Kozak sequence (consensus: A/GXXAUGG) The eukaryotic mRNA 5’ cap and 3’ poly(A) tail are also involved in initiation Scanning model for eukaryotic initiation The cap is bound by eIF4E and the tail is bound by poly(A) binding protein (PABP). These interactions stabilize interaction with eIF4G, forming a closed loop complex, which recruits eIF4A/eif4B that promotes unwinding of mRNA to aid in efficient ribosome scanning The circular feature of RNA acts as quality control feature, distinguishing between competent and incompetent mRNA substrates Most other initiation factors (eIF1, 1A, 2, 3, 5) prime the 40S subunit Translation I Side notes: for scanning. Similar or bacterial IF1 and IF3, eIF1A binds of the A site and eIF1 positions close to the E site, and together, guide initiator tRNA to P site To do so, the initiator tRNA is bound to a protein eIF2 in a ternary complex with GTP. Together, the small ribosome subunit, eIF2 ternary complex, and mRNA form the pre-initiation complex (PIC) The pre-initiation complex is now ready for scanning The scanning ribosome moves along the mRNA and identifies the start codon AUG (aided by the Kozak sequence) Start codon recognition triggers irreversible release of eIF1 and inorganic phosphate from eIF2 Similar to bacterial IF2, eukaryotic eIF5B catalyzes joining of the large subunit through GTP hydrolysis (subunit joining) Once the large subunit joins and initiation factors have dissociated, ribosome is now ready to begin elongation!