4# Translation mechanisms

• Lecture is first in a two-part series.
• Focus is basic molecular mechanisms of translation.
• The second lecture covers global and individual mRNA translation regulation.
• Mechanisms apply to eukaryotic translation, prokaryotic translation is discussed in detail.

Translation and the Genetic Code

• Translation converts the genetic information encoded in mRNA into a polypeptide sequence (protein).
• DNA → mRNA (transcription) → Protein (translation) in the central dogma of molecular biology.

Directionality of Translation

• mRNA is read in the 5'→ 3' direction.
• Proteins are synthesized in the N-terminal → C-terminal direction.

The Genetic Code

• The genetic code links the mRNA sequence to the final amino acid sequence in the protein.
• Properties of the genetic code:
• Collinear: The sequence of bases in mRNA corresponds directly to the amino acid sequence in the protein.
• Non-overlapping: Each triplet codon is read separately, without overlap.
• Triplet (Codon-based): Each three-nucleotide codon specifies a single amino acid.
• Triplet nature of code exists because there are 4 nucleotides (A, U, G, C) in RNA but 20 amino acids to be encoded.

Translation as a Key Process

• Translation is the active process of protein synthesis.
• It connects the mRNA life cycle and the protein life cycle.
• mRNA serves as the template for protein synthesis.
• Newly synthesized proteins emerge from translation and become functional in the cell.

Correlation Between mRNA and Protein Levels

• mRNA levels and protein levels are often correlated because translation converts mRNA into protein.
• Translation regulation affects protein output.
• Can occur across the entire cell's transcriptome and proteome, globally.
• Can occur at the individual mRNA level, thus mRNAs may be more or less efficiently translated.
• Regulatory mechanisms are to be explored in detail in the next lecture.

Takeaway

• Translation process decodes mRNA into proteins.
• Occurs in the 5'→ 3' direction in mRNA and N-terminal → C-terminal direction in proteins.
• The genetic code is collinear, non-overlapping, and triplet-based.
• Translation connects mRNA and protein life cycles, with correlation between mRNA and protein levels.
• Regulation of translation occurs at both a global and individual mRNA level and will be the focus of the next lecture.

Overview of the Translation Process

• There are three phases: initiation, elongation, and termination.
• Each phase involves specific molecular interactions and dedicated protein factors that ensure accurate and efficient protein synthesis.

The Three Phases of Translation: Initiation

• Process where all required molecular components assemble at the start codon of the mRNA.
• Key initiation steps:
• The small ribosomal subunit binds to the mRNA.
• Initiation factors assist in correctly positioning the ribosome.
• The start codon (AUG) is recognized, and the large ribosomal subunit joins the complex.
• Translation is ready to begin.

Elongation

• Elongation involves repeated cycles of amino acid incorporation into the growing polypeptide chain.
• Consists of decoding, the correct charged aminoacyl-tRNA binds to the ribosome.
• Peptide bond formation is catalyzed by the ribosome, linking new amino acids.
• Translocation, the ribosome moves to the next codon (triplet) in the mRNA.

Termination

• Termination occurs when the ribosome encounters a stop codon.
• Steps of Termination:
• Release factors recognize the stop codon.
• The completed polypeptide is released from the ribosome.
• The ribosome dissociates into its subunits, and components are recycled for the next translation cycle.

Components Required for Translation

• mRNA contains the open reading frame (ORF), including the start and stop codons.
• Ribosome: Consists of large and small subunits.
• Aminoacyl-tRNAs: Transfer RNAs (tRNAs) charged with the correct amino acids.
• Protein factors:
• Initiation factors (IFs or eIFs in eukaryotes)
• Elongation factors (EFs or eEFs in eukaryotes)
• Release factors (RFs or eRFs in eukaryotes)
• Many translation factors function as GTPases, hydrolyzing GTP → GDP.
• Ensures irreversible steps in translation and regulates the process.

Structural Features of mRNA and Their Role in Translation

• Eukaryotic mRNA has an open reading frame (ORF) that encodes the protein sequence.
• It has start and stop codons, defining the beginning and ending of translation.
• Consists of Untranslated Regions (UTRs).
• 5' UTR: Upstream region, involved in translation regulation.
• 3' UTR: Downstream region, plays roles in mRNA stability and translation efficiency.

Terminal Features Required for Efficient Translation

• 5' Cap (7-methylguanosine cap) has a chemical modification at the 5' end of the mRNA.
• Functions:
• Protects mRNA from degradation.
• Recognized by the initiation factor eIF4E, which helps recruit the ribosome.
• 3' Poly(A) Tail, a string of adenine nucleotides added to the 3' end of mRNA.
• Functions:
• Protects mRNA from degradation.
• Binds to poly(A)-binding protein (PABP), which interacts with translation machinery to enhance efficiency.
• 5' and 3' UTRS can influence translation efficiency, mRNA stability, and localization within the cell.
• Regulatory sequences in UTRs, called cis-acting elements, are recognized by RNA-binding proteins to control translation.

Comparison of Prokaryotic and Eukaryotic Translation: Structural Differences in mRNA

• Eukaryotic mRNA includes a 5' cap and 3' poly(A) Tail, both absent in Prokaryotic mRNA.
• Prokaryotic mRNA requires the Shine-Dalgarno Sequence for start codon recognition.
• Eukaryotic mRNA uses the 5' cap and scanning mechanism.

Initiation Mechanisms

• Prokaryotic initiation:
• Uses upstream ribosome binding site of the Shine-Dalgarno Sequence.
• This sequence base-pairs with the 16S rRNA of the small ribosomal subunit to position the ribosome correctly.
• Eukaryotic initiation:
• No Shine-Dalgarno Sequence.
• The 5' cap recruits initiation factors, which help load the small ribosomal subunit.
• The ribosome scans the 5' UTR for the first AUG codon in an optimal Kozak sequence context.

Summary

• Translation occurs in three main phases: initiation, elongation, and termination.
• Key components include mRNA, ribosomes, tRNAs, and specialized protein factors.
• Eukaryotic translation requires the 5' cap and 3' poly(A) tail, prokaryotic translation relies on the Shine-Dalgarno sequence.
• GTPases regulate translation by ensuring key steps are irreversible.
• UTRs and RNA-binding proteins play significant roles in translation regulation.

Transfer RNA (tRNA) – The Adapter Molecule - Role of tRNA in Translation

• tRNAs act as physical adapters, linking the codon with triplet sequence in mRNA to the proper amino acid.
• Each tRNA is covalently linked to an amino acid at its 3' end.
• The 3' hydroxyl group of tRNA is bound to the amino acid, then incorporated into the growing polypeptide chain.

Structure of tRNA

• tRNA has a cloverleaf secondary structure with four loops and stems and an L-shaped tertiary structure formed by additional interactions.
• Two key functional sites:
• 3' End (Acceptor Arm): Carries the amino acid.
• Anticodon Loop: Contains the three-nucleotide anticodon that base-pairs with the mRNA codon.

tRNA Decoding and the Wobble Hypothesis

• Genetic code is degenerate, meaning multiple codons can specify the same amino acid.
• Wobble base pairing at the third codon position allows flexibility in codon recognition.
• A G in the anticodon can pair with C or U in the codon.
• Some tRNAs contain inosine (I), a modified adenine, which can pair with A, U, or C.
• Significance:
• Reduces the number of required tRNA molecules (30-50 tRNAs per organism instead of 61).
• Increases efficiency of translation.

The Ribosome

• Ribosomes are large macromolecular complexes made of rRNA and ribosomal proteins.
• Consist of two subunits:
• Small subunit interacts with mRNA and helps decode the genetic information.
• Large subunit catalyzes peptide bond formation.
• rRNA makes up ~2/3 of ribosome mass and forms complex secondary and tertiary structures.

Functions of Ribosomal Subunits

• Small Ribosomal Subunit mediates interactions with mRNA and contains the decoding center.
• Large Ribosomal Subunit contains the peptidyl transferase center, which catalyzes peptide bond formation.

Ribosome Binding Sites for tRNA

• The ribosome has three distinct tRNA binding sites
• A-site (Aminoacyl site) accepts new aminoacyl-tRNA, bringing the next amino acid.
• P-site (Peptidyl site) holds the growing polypeptide chain and facilitates peptide bond formation.
• E-site (Exit site) holds the deacylated tRNA before it exits the ribosome.

Translocation Process

• During elongation, the ribosome moves along the mRNA, shifting tRNAs through the binding sites.
• After peptide bond formation:
• The P-site tRNA moves to the E-site (exit site).
• The A-site tRNA moves into the P-site.
• The A-site is now free to accept a new aminoacyl-tRNA, and the cycle continues.

Summary

• tRNA serves as an adapter molecule, linking mRNA codons to their corresponding amino acids.
• Wobble base pairing.
• Enabled, the genetic code degeneracy has ribosomes as a molecular machine responsible for protein synthesis, with a small and large subunit.
• The ribosome has three binding sites (A, P, and E sites).
• Peptide bond formation occurs in the peptidyl transferase center of the large subunit.
• During translocation, tRNAs shift between the A, P, and E sites allowing the ribosome to move along the mRNA.

Overview of Translation Mechanisms

• Consists of three main steps:
• Initiation, consisting of the Assembly of ribosomal subunits and loading of the initiator tRNA.
• Elongation, consisting of the addition of amino acids to the growing polypeptide chain.
• Termination, consisting of the complexed polypeptide and ribosome disassembly.
• Translation principles are similar with differences in the initiation mechanisms between prokaryotes and eukaryotes.

Translation Initiation in Prokaryotes

• Goal of the initiation process is to load a charged initiator tRNA into the P-site to pair with the AUG start codon.
• This requires a small ribosomal subunit (30S) which binds to mRNA; Initiation factors (IFs) which assist in assembly; and a large ribosomal subunit (50S) which joins to form the full 70S initiation complex.

Role of the Shine-Dalgarno Sequence

• A few nucleotides upstream of the AUG start codon in mRNA.
• Base-pairs with the 16S rRNA in the small ribosomal subunit, correctly positioning the ribosome.
• Important in polycistronic mRNAs.

The Initiator tRNA (tRNAi^fMet)

• Specialized initiator tRNA used only for translation initiation.
• Carries N-formylmethionine (fMet) instead of a regular methionine.
• Binds directly to the P-site unlike other tRNAs.
• Requires Initiation Factor 2 (IF2), a GTPase, to assist in binding.
• Organisms contain two types of methionine tRNAs:
• Initiator tRNA (tRNAi^fMet) – Used exclusively for initiation.
• Elongator tRNA (tRNA^Met) – Used for adding methionine in the middle of a protein chain.

Role of GTPases in Translation

• Many translation factors (e.g., IF2) are GTP-binding proteins (GTPases).
• These proteins switch between active (GTP-bound) and inactive (GDP-bound) states.
• Hydrolysis of GTP → GDP causes a conformational change that regulates activity.

Regulation of GTPases

• GTPase-Activating Proteins (GAPs) stimulate GTP hydrolysis, converting GTP-bound proteins to inactive GDP-bound form.
• Guanine Nucleotide Exchange Factors (GEFs) facilitate GDP-GTP exchange, activating the GTPase again.
• GTPases regulate translation initiation, elongation, and termination.
• Similar GTPase mechanisms are also found in cell signaling pathways (e.g., G-proteins and GPCRs).

Step-by-Step Mechanism of Prokaryotic Translation Initiation

• Free ribosomes exist in a dynamic equilibrium between assembled and dissociated states.
• The 30S ribosomal subunit binds to Initiation Factors IF1 and IF3, which prevent premature binding of the 50S subunit.
• The mRNA binds to the 30S subunit, with the Shine-Dalgarno sequence positioning the AUG start codon in the P-site.
• Initiator tRNA (tRNAi^fMet) binds to the AUG start codon in the P-site.
• IF2-GTP assists in bringing tRNAi^fMet to the ribosome.
• The 50S large ribosomal subunit joins the complex.
• Triggering, GTP hydrolysis by IF2, leading to:
• Release of IF1, IF2, and IF3.
• Formation of the complete 70S ribosome ready to elongation.

Summary

• Translation initiation requires the Shine-Dalgarno sequence to correctly position the ribosome.
• Initiation factors and specialized initiator tRNA carrying N-formylmethionine needed. GTPase to facilitate initiator tRNA binding and ribosome assembly.
• GTP hydrolysis completes the initiation process, with the fully assembled 70S ribosome ready for elongation.

Overview of Elongation in Prokaryotic Translation

• Elongation is a cyclic process which has three key steps:
• tRNA Loading, new aminoacyl-tRNA enters the A-site of the ribosome.
• Peptide Bond Formation, the growing polypeptide chain is transferred from the P-site tRNA to the A-site tRNA.
• Translocation, in which the ribosome moves one codon forward, shifting the tRNAs into their new positions.
• Elongation requires three elongation factors (EFs):
• EF-Tu (GTPase) delivers aminoacyl-tRNA to the A-site.
• EF-Ts regenerates EF-Tu by exchanging GDP for GTP.
• EF-G (GTPase) facilitates translocation of the ribosome.

tRNA Loading into the A-Site

• The aminoacyl-tRNA is delivered to the A-site as part of a complex with EF-Tu-GTP.
• When the anticodon-codon pairing is correct:
• GTP hydrolysis occurs → EF-Tu is released from the ribosome.
• The A-site tRNA remains bound, ready for peptide bond formation.
• When the anticodon-codon pairing is incorrect:
• The tRNA dissociates before GTP hydrolysis can take place (kinetic proofreading). -Ensuring high fidelity of translation.

Peptide Bond Formation

• The amino group of the A-site amino acid attacks the ester bond linking the polypeptide to the P-site tRNA.
• The peptidyl transferase center of the ribosome catalyses the reaction.
• The growing polypeptide chain is transferred from the P-site tRNA to the A-site tRNA.
• Catalyzed entirely by ribosomal RNA (rRNA), the ribosome is a ribozyme.

Ribosome Translocation

• EF-G-GTP binds to the ribosome, causing a conformational change.
• GTP hydrolysis occurs, thus energy is released.
• This drives the ribosome to move along the mRNA by one codon:
• The A-site tRNA moves to the P-site; the P-site tRNA moves to the E-site and is ejected.
• The A-site is now empty, ready for the next aminoacyl-tRNA.
• GTP hydrolysis:
• Ensures directionality of translocation and is irreversible.

Ribosomal Structure and the Role of rRNA in Peptide Bond Formation

• The ribosome's catalytic activity is due to rRNA, not ribosomal proteins.
• The peptidyl transferase center is located in the large ribosomal subunit (50S).
• The N3 nitrogen of an adenine in the 23S rRNA plays a key role in activating the amino group for peptide bond formation.
• The discovery that ribosome is an RNA-based enzyme or ribozyme was confirmed.

The Polypeptide Exit Tunnel

• As elongation proceeds, the growing polypeptide chain exits through a tunnel in the large ribosomal subunit:
• Guiding the nascent protein out of the ribosome and helps with proper protein folding before release.

Structural Mimicry of Elongation Factors

• EF-Tu-tRNA and EF-G have similar structural shapes.
• This allows EF-G to fit into the A-site, mimicking a tRNA during translocation.
• Molecular mimicry is essential for efficient ribosome function.

Summary of Prokaryotic Translation Elongation

• Aminoacyl-tRNA is delivered to the A-site by EF-Tu-GTP
• Correct codon-anticodon pairing triggers GTP hydrolysis, releasing EF-Tu.
• Peptide bond formation occurs, catalyzed by rRNA (a ribozyme).
• Translocation is driven by EF-G-GTP, shifting the ribosome forward by one codon.
• The process repeats until a stop codon is encountered in the mRNA.

Overview of Translation Termination

• Termination occurs when the ribosome encounters a stop codon in the mRNA:
• UAG ("Amber), UAA ("Ochre"), and UGA ("Opal")
• Because no tRNAs recognize code sequence of stop codons, release factors (RFs) bind to the ribosome to trigger termination.

Role of Release Factors in Termination

• Release Factors (RFs) are specialized proteins that recognize stop codons through direct RNA-protein interactions.
• Prokaryotes have two primary release factors, RF1 which recognises UAG and UAA and RF2 which recognises UGA and UAA.
• These release factors bind to the A-site of the ribosome when a stop codon is present.
• This triggers the recruitment of RF3.
• RF3 hydrolyzes GTP
• Peptide is cleaved from the P-site tRNA with dissociation of release factors. Then the ribosome disassembles for a new round of translation.

Summary of Prokaryotic Translation Termination

• Termination occurs when a stop codon reaches the A-site which is recognized by the release factors (RF1 and RF2) that bind to the ribosome.
• RF3 hydrolyzes GTP, triggering polypeptide release and ribosome disassembly.
• The ribosome separates into its subunits, ready for the next round of translation.

Polysomes and Simultaneous Translation

• During active translation, multiple ribosomes can translate a single mRNA simultaneously.
• These complexes are called polysomes, possible due to the co-linearity of the genetic code.
• Translation starts at the AUG start codon and continues to the stop codon.
• Multiple ribosomes can attach and move along the mRNA without interfering with one another.
• This translates more proteins and increases the efficiency of protein synthesis.

Eukaryotic VS. Prokaryotic Translation

• Major differences is in initiation mechanisms.
• Prokaryotes use the Shine-Dalgarno sequence to position the ribosome.
• Eukaryotes use the 5' Cap and scanning mechanism to locate the start codon.
• There are differences in the ribosome.
• Prokaryotes range 70S where Eukaryotes are 80S.
• Prokaryotes: No poly(A) Tail where Eukaryotes: multiple
• Initiation Factors: Prokaryotes like IF1 and EF2 where Eukaryotes have many eIFs.

Eukaryotic Translation Initiation Mechanism

• Eukaryotic ribosomes come in 80S.
• A 40S small subunit containing the decoding center, and a 60S large subunit containing peptidyl transferase center.
• Large subunit contains three rRNAs instead of two, consisting of 28S, 5S, and 5.8S rRNAs.
• Key eIFs complex in initiation, which is eIF4F Complex. Consisting of 5' Cap which is bound EIF4E.

Step-by-Step Mechanism of Initiation in Eukaryotes

• First, a 43S pre-initiation complex which has of eIF1, eIF1A, and eIF3, that bind to the 40S ribosomal subunit. eIF2-GTP loads the initiator Met-tRNAi into the P-site.
• Then, mRNA recogition. The complex now binds EIF4F which is the 5' Cap.
• Once the mRNA is unwound, then the complex finds the initiation sequence or what they call start codon.
• When the AUG start codon is found, the eIF2-GTP hydrolyzes GDP, triggering the release.

The Poly(A) Tails role in translation

• eIF4G binds to the Poly(A)-Binding Protein (PАВР), from the eIF4F complex.
• This circulates the mRNA, by enhancing efficient translation.
• This means only mRNA with both a 5' Cap and Poly(A) are translated.

The Role of elF3 in translation

• eIF3 is a Multi subunit protein which can recruit more subunits to aid in small Rinosomes and is critical for initial interaction.
• Recent discovery states the eIF3D subunits can bind independently to the Cap. Proving that initiation can still occur.

Keys Summary and Steps of Translation for Eukaryotes

• Eukaryotes lack the Shine-Dalgarno Sequences, so ribosomes scans and have higher number of IFs.
• The steps are
• One. Formation of 43 pre-initiation
• Two. Biding of eLF4F
• Three.Scanes for sequence
• Four. start codon
• Five. Bind 60s subnit.
• Polysomes allow for translation to increase with the interaction of poly(A) tails. There for the key is recognition since the step allows for regulation of protein production.

Eukaryotic Elongation and Termination

• Have High similarities with the previous models. There fore their are direct counter parts.
• With Key Differences such as having Eukaryotes having only one release factor vs many in prok.

Differences Between Prokaryotic and Eukaryotic

• Eukaryotes are separated translation trans in nucl. Trans out.
• initiation. Uses Shine-Dalgarno Prok scans.
• Variable Stability high stable mRNA,
• Have more of short and open frame work.
• E and Term are the Same.
• Key is scanning method instead of Shine Dar.
• and mRNA need exports to preform properly

Final thouts

• elongation and are identical because euka key to regulation in translation. Proks are highly effective dwe ti co trans, and have euka for more reg.

Translation: Accuracy in Translation

• 10–20 amino acids synthesized per second, high fidelity due to multiple fidelity checks.
• Most translation errors occur at the ribosome because of an incorrect tRNA selection in the A-site.

The Ribosome in Translation Fidelity

• Enhances by monitoring base-pairing between codon and anticodon, structural changes in decoding for binding, use kinetic reading to correct, as well as reject.
• A decoding center and structure changes in sub units can change once TRnA binds. These induce correct binding.

Kinetic Proofreading in Translation Elongation

• Time depended error to correct.
• Translation controlled by these steps. TRNA and accomodate.

Step 1: Reading Before Hyrdo

• EF delivers the TRna and if correct the protes
• If not that causes seperation

coupling

• By using ef and his bind together.

summary

• Multiple fidel checks prevent errors.

Final Points

• Kinectic helps prevent mistakes.
• Ribosomes are Active.

Summary of Translation

• Three main stages.
• Intitation. Assemble.
• Elon Sequential Addtion .
• Term releases

Key Comp

• MRN code, TRNA code, Ribosomes is to.
• Factors. Help assembly

fidelity

• Translation fast but 10-20 sec

Step

• Trna, remove inc and do not react corectly.

Sum final

• Highly Reg is muti step inc ter and trans. are all.
• fidelity of tran is by the prodfing help by text books and review.