4.6 RNA Translation

Translation initiation steps:

(1) Met-tRNAi Met Priming (2) Met-tRNAi Met Transport (3) mRNA Packaging (4) mRNA Attachment (5) mRNA Scanning (6) Initiation Codon Recognition (7) Ribosomal Subunit Association (8) Initiation Complex Formation

(3) Translocation

(1) The newly synthesized peptidyl-tRNA @ the A-site must be translocated to the P-site (2) The uncharged or deacylated tRNA already occupying the P-site must be transferred to E-site—so as to not only effect its release from the ribosome but also to make way for the incoming peptidyl-tRNA, thereby vacating the A-site (3) In sync with the above two events, the ribosome must also advance exactly three nucleotides forward along the mRNA track in order to accommodate the next aa-tRNA

Met-tRNAi Met Priming

- AUG initiation codon is recognized by the so-called initiator tRNA covalently attached to its cognate Met residue (methionyl-RNAiMet)—virtually all eukaryotic proteins begin with Met residue though this is sometimes cleaved off during post-translational modification (PTM) - In order to recognize the AUG initiation codon, Met RNA is first assembled into a ternary complex with GTP-bound eIF2 so as to prepare or "prime" it for transport to the ribosome - The GTP-eIF2-metRNAiMet complex subsequently translocates to the P-site within the 40S subunit of ribosome so as to transport the metRNAiMet in order to allow it to base pair (via its CAU anticodon) with the AUG initiation codon within mRNA - The ability of methionyl-tRNAiMet to recognize the AUG initiation codon arises from its ability to specifically bind to eIF2 and the P-site within the 40S subunit of ribosome—on the other hand, methionyl-tRNAMet that recognizes AUG internal codons can only be accommodated within the A- site thus barring it from initiating protein synthesis

(8) Initiation Complex Formation

- Association of 60S subunit with 40S subunit within the p80S complex triggers eIF5B-catalyzed hydrolysis of its bound GTP to GDP - This event releases both eIF5b and eIF1A from the p80S complex coupled with a conformational change that ultimately generates the 80S complex - The 80S complex is fully "primed" to accept/recruit an incoming aminoacyl-tRNA at the A-site in order to "stitch" its aminoacyl group onto the C-terminal of methionine within the methionyl- tRNAiMet waiting @ the P-site—the proto-peptidyl-tRNA (proto -> mini | primitive | initial) - At this stage, the ribosome bids an "emotional" farewell to eIFs and welcomes aboard the eukaryotic elongation factors (eEFs) to begin the next stage of its journey along the mRNA track—enter translation elongation!

(7) Ribosomal Subunit Association

- Binding of GTP-bound eIF5B to the 48S complex triggers eIF2-catalyzed hydrolysis of its bound GTP to GDP —an event that results in the release of all eIFs but eIF1A - The release of eIFs from the 48S initiation complex makes way for the docking of the 60S subunit—which binds to the same interface on 40S subunit that had earlier "piggybacked" the eIFs - The association of 60S subunit with the 48S complex generates the pre-80S (p80S) complex

Translation Elongation: (1) Decoding

- Coupled with GTP hydrolysis, the incoming aminoacyl-tRNA (aa-tRNA) is escorted/delivered by eEF1A and eEF1B to the A-site within the ribosome - In this manner, the ribosome selects and recruits an aa-tRNA whose anticodon is complementary to the mRNA codon @ the A-site in a phenomenon referred to as "decoding" - eEF1A and eEF1B specifically bind to tRNAs only after they become aminoacylated—ie aa-tRNAs

(2) Transpeptidation

- Peptidyl transferase—an RNA component of ribosome (ribozyme)—catalyzes the formation of a peptide bond through the nucleophilic displacement of the peptidyl- tRNA @ the P-site by the aminoacyl group of aa-tRNA @ the A-site in a reaction referred to as "transpeptidation" - This reaction thus not only increases the peptide length of the peptidyl-tRNA from n to n+1 but also moves it to the A-site - The release of free energy upon the cleavage of "high- energy" ester linkage in peptidyl-tRNA drives the reaction to completion

(2) Met-tRNAiMet Transport

- Ribosome undergoes a constant recycling process whereby it assembles prior to initiation of each round of polypeptide synthesis that terminates with the dissociation of ribosomal subunits - Prior to the formation of PIC, the separated 40S subunit of ribosome recruits eIF1, eIF1A and eIF3 (and presumably eIF5) - The 40S-eIFs complex subsequently serves as a docking platform for the recruitment of GTP-eIF2- metRNAiMet complex resulting in the formation of 43S complex (or PIC) - Within the 43S complex, methionyl-tRNAiMet binds to the P-site so as to essentially serve as a proto-peptidyl-tRNA—thus being in a position to receive the second amino acid from the next aa- tRNA as dictated by the mRNA codon following the initiating AUG codon

(6) Initiation Codon Recognition

- The AUG initiation codon (or TIS) of mRNA is embedded within the GCC(A/G)CCAUGG consensus sequence - While the eIFs play a central role in guiding the 40S ribosomal unit to the initiation codon, its ultimate recognition is dependent upon the ability of methionyl-tRNAiMet to base pair (via its CAU anticodon) with the AUG codon within mRNA - Such codon-anticodon base pairing induces a conformational change within the p48S complex to generate the 48S complex

aminoacyl-tRNA

- The covalent attachment or esterification of a tRNA with a specific amino acid (aa) for which it harbors an anticodon generates the corresponding aminoacyl-tRNA (aa-tRNA) conjugate in a process referred to as "aminoacylation" - What is the difference between tRNA^Arg and arginyl-tRNA? The tRNA^Arg is the uncharged (free) tRNA that has the capacity to bind Arg, whereas arginyl-tRNA is the tRNA^Arg in which Arg is covalently attached to its 3'- OH group—strictly, Arginyl-tRNA should be written as "arginyl- tRNA^Arg"

Wobble-hypothesis

- The site of mRNA codon degeneracy at the third position is exquisitely mirrored by anticodons in tRNAs in that the latter harbor a modified base such as inosine (I) at the complementary position— ie the 5'-nucleotide in tRNA anticodon that base pairs with the 3'-nucleotide (the third position) in mRNA codon - non-Watson-Crick base pairing between tRNA and mRNA allows a greater degree of conformational freedom (at the third position of the codon) needed to accommodate the same tRNA within degenerate codons—such non-canonical pairing is referred to as "Wobble base pairing"

Ribosome: peptidyl transferase

- The task of removing the aminoacyl group of aminoacyl-tRNA and adding it to a nascent polypeptide chain falls to an RNA component (28S) of 60S subunit called "peptidyl transferase" - Peptidyl transferase is a ribozyme—an exclusively RNA-based catalyst (or a ribonucleic acid enzyme) Peptidyl transferase promotes nucleophilic attack of the incoming amino group of aminoacyl-tRNA (A-site) on the carbonyl group of peptidyl-RNA (P-site), thereby resulting in the: (1) displacement of the tRNA of the peptidyl-tRNA (harboring n amino acids) (2) formation of a new peptide bond with the incoming amino acid (transpeptidation) (3) transfer of nascent polypeptide chain harboring n+1 amino acids to A-site (4) translocation of uncharged/deacylated tRNA from P-site to E-site (5) translocation of the new peptidyl-tRNA (harboring n+1 amino acids) to P-site

Codons

- Triplets of bases that code for a specific amino acid, start, or stop codon - Codons are decoded by tRNA - since there are 4 nucleotides, there are 4^3 = 64 codons. These 64 codons are known as the genetic code. - of 64 codons, 61 code for amino acids, 3 are nonsense, or stop codons - Stop codons: UAA, UAG, UGA - Start codons/translation initiation site (TIS): AUG (also methionine, most common), UGG (also tryptophan)

Translation Termination: (1) Polypeptide Release

- When the ribosome encounters one of the three stop codons—UAA, UGA or UAG—at the A-site, it comes to a grinding halt due to the fact there is no aa-tRNA counterpart that can base pair with one of these stop codons in mRNA - Such halting signals the loading of eukaryotic release factors (eRFs) such as eRF1, eRF3 and Rli1—also called ABCE1 - Upon recognition and confirmation of one of the stop codons at the A-site within the ribosome, eRF1 triggers the hydrolysis of the ester bond in peptidyl- tRNA @ the P-site—thereby releasing the newly synthesized polypeptide - Upon the cleavage of the ester bond, GDP-bound eRF3 loads onto the ribosome to accelerate the release of the cleaved polypeptide

Translation of tRNA codons

- a specific tRNA—carrying a cognate amino acid—base pairs via its anticodon with a corresponding mRNA codon - In so doing, tRNAs transfer or transport their cognate amino acids to a nascent polypeptide chain guided by the ribosomal translational machinery

Translational Machinery (ribosome)

- consists of small and large subunit, translates mRNA into polypeptide - reads the mRNA in a 5' -> 3' direction, from N to C terminus Translation can be divided into 3 major steps: (1) Initiation—requires eukaryotic initiation factors (eIFs) (2) Elongation—requires eukaryotic elongation factors (eEFs) (3) Termination—requires eukaryotic release factors (eRFs) - Multiple ribosomes can bind to a single mRNA transcript adopting a "beads- on-a-string" appearance called "polyribosome" or "polysome"

(2) Ribosomal Recycling

- eRF3 undergoes GDP-GTP exchange in order to induce a conformational change within the ribosome so as to facilitate the release of eRF1 - Upon the release of eRF1, Rli loads on to the ribosome and, in so doing, displaces GTP- bound eRF3 - Next, Rli facilitates the release of bound mRNA and deacylated tRNA from the ribosome - The release of mRNA and tRNA finally triggers the dissociation of ribosome into constituent 40s and 60s subunits so that they can be recycled for another round of translation

Codon promiscuity

- many tRNAs can base pair with more than one mRNA codon—eg tRNAAla harboring the IGC anticodon recognizes three (GCA/GCC/GCU) of the four mRNA codons for alanine (the fourth codon being GCG) - Thus, the actual number of tRNAs required for productive protein synthesis is much less than 61—but a minimum of 32 tRNAs are required to recognize all 61 mRNA codons according to "Wobble hypothesis"—vide infra - Paradoxically, the genomes of most eukaryotes encode over 100 tRNAs—many of these tRNAs encode non-standard amino acids while others are non-functional or represent pseudo-genes

tRNA: 2D cloverleaf

- tRNA is a polymer (~ 80nt long) comprised of three stem-loops and a solo stem called the stem "acceptor stem" - One of the three stem-loops within tRNA harbors a trinucleotide sequence that is complimentary to one of the mRNA codons—such a complimentary triplet of bases within tRNA is referred to as the "anticodon", the corresponding stem-loop the "anticodon arm" - On the other hand, the 3'-OH end of the acceptor stem of tRNA covalently attaches via an ester linkage to one of the 20 amino acids in a highly specific manner - For each of the 20 amino acids, there exists at least one cognate tRNA harboring an anticodon to match the corresponding codon in mRNA - The various secondary structural motifs constituting the 2D cloverleaf form of tRNA fold into a complex 3D structure resembling the letter L—such 3D shape is stabilized by extensive stacking interactions as well as non-Watson-Crick base pairing between bases in various stems - The various secondary structural motifs constituting the 2D cloverleaf form of tRNA fold into a complex 3D structure resembling the letter L—such 3D shape is stabilized by extensive stacking interactions as well as non-Watson-Crick base pairing between bases in various stems

(3) mRNA Packaging

- the first AUG codon immediately following the 5'-UTR sequence serves as the initiation codon or translation initiation site (TIS) - The AUG initiation codon, or TIS, is embedded within the GCC(A/G)CCAUGG consensus sequence—a motif that is recognized by the binding of various eIFs

(4) mRNA Attachment

After the preparation of mRNA with various eIFs, the mRNA-eIFs complex is shuttled to the ribosome, where it attaches to the 43S complex pre-loaded with metRNAiMet to form the pre-48S (p48S) complex

(5) mRNA Scanning

In sync with the assembly of the p48S complex, the 40S ribosome begins to scan the mRNA transcript downstream of the 5'-cap to locate the AUG initiation codon - Such mRNA scanning requires the cooperation of all parties including, in particular, the attached eIFs, albeit to varying degrees—eg omission of eIF1A mitigates the scanning process, while the lack of eIF1 completely halts/abrogates it

Aminoacyl-tRNA Synthetase (aaRS)

Synthesis of aminoacyl-tRNA (aa-tRNA) is driven by aminoacyl-tRNA synthetase (aaRS) via the following two steps: (1) Activation of a specific amino acid (aa) with ATP to generate the "high-energy" aminoacyl-adenylate (aa-AMP) intermediate: aa + ATP <=> aa-AMP + PPi (2) The aa-AMP intermediate subsequently reacts with a cognate tRNA (ie one that harbors the anticodon for the specific aa to be covalently attached): aa-AMP + tRNA <=> aa-tRNA + AMP - In this manner, aaRS-catalyzed synthesis of aa-tRNA ensures that it harbors the correct anticodon for the amino acid it is carrying—aaRS thus serves as a "matchmaker" in that it matches a specific amino acid with its anticodon within the cognate tRNA by virtue of its ability to recognize their 3D structures!

Ribosome: sedimentation coefficient

in Eukaryotes: (1) 60S large subunit—comprised of three RNA polynucleotides (28S/5.8S/5S) and ~49 polypeptides (2) 40S small subunit—comprised of a single RNA polynucleotide (18S) and ~33 polypeptides Total: 80S

tRNA binding sites:

the ribosome harbors three distinct sites for simultaneously accommodating three tRNAs at various stages: (1) A-site for aminoacyl-tRNA (the incoming tRNA carrying an Amino acid) (2) P-site for peptidyl-tRNA (the tRNA attached to the nascent Polypeptide) (3) E-site for deacylated tRNA (the tRNA about to Exit after donating its amino acid)