220 - lecture 17

Central Dogma IV: Translation (Part 2) Elongation & Termination

Overview

  • Interpretation of the genetic code into proteins.

  • Functionality of tRNAs and ribosomes in translation.

  • Main stages of translation and the required energy.

  • Influence of mutations and antibiotics on translation.

Decoding the Codons: The Role of tRNAs

  • tRNAs serve as adaptors between mRNA codons and specific amino acids.

  • Each tRNA transports one amino acid and recognizes mRNA codons via its anticodon.

    • The anticodon pairs with the complementary mRNA codon during translation.

  • tRNA Structure:

    • Length: 73–93 nucleotides.

    • 2D structure: cloverleaf; 3D structure: L-shaped.

    • Key regions include:

    • Amino acid acceptor arm (3′ CCA): This region binds to the amino acid.

    • Anticodon loop: Binds to the codon in mRNA.

    • Contains modified nucleotides to stabilize folding.

  • Wobble Hypothesis:

    • Some tRNAs can pair with more than one codon due to flexible base pairing at the third codon position.

tRNA Charging: Attaching Amino Acids to tRNAs

  • Aminoacyl-tRNA synthetases attach the appropriate amino acid to tRNA at its 3′ end.

  • The charging process requires ATP, forming a high-energy bond utilized in peptide bond formation.

  • Each amino acid corresponds to a specific synthetase enzyme.

  • Enzyme proofreading corrects any mismatched amino acid-tRNA pairs.

Initiator tRNA

  • Protein synthesis starts with methionine as the first amino acid (N-terminal).

    • In bacteria, this is a modified version called formyl-methionine (fMet).

  • Methionine is unique as it can bind to the P site of the ribosome before the large ribosomal subunit attaches.

  • The methionine may be removed by proteases after translation initiation.

  • Key Idea: Initiator tRNA establishes the start codon and reading frame for translation.

Summary: The tRNA Connection

  • tRNAs are essential for translating codons into amino acids.

  • Accuracy relies on proper amino acid charging and codon-anticodon pairing.

  • Initiator tRNA is critical in determining the reading frame for translation.

Translation Overview

  • Translation: Process of decoding mRNA to synthesize a polypeptide.

  • Consists of three primary stages:

    • Initiation: Ribosome assembles on the mRNA and locates the start codon.

    • Elongation: Series of amino acids are sequentially added as the ribosome traverses the mRNA.

    • Termination: Encountering a stop codon leads to the release of the completed polypeptide.

  • Requires GTP and multiple protein factors including initiation, elongation, and termination factors.

  • Takes place on ribosomes in the cytoplasm or on rough endoplasmic reticulum (RER) for proteins destined for secretion.

Initiation of Translation in Eukaryotes

  • Ribosomal subunits assemble on the mRNA in a layered configuration:

    • Small subunit (40S): Decodes genetic messages.

    • Large subunit (60S): Catalyzes peptide bond formation.

  • The initiation process is intricate and involves multiple steps:

    • Requires at least 12 initiation factors (totaling over 25 polypeptides).

    • mRNA binds to the small subunit first, forming the 43S complex.

    • The 43S complex scans for the AUG start codon, forming the 48S complex when aligning with the mRNA's 5' end.

    • After the initiation factors disband, the large subunit binds to complete the assembly of the 80S ribosome ready for elongation.

Initiation of Translation in Eukaryotes (Continued)

  • Initialization Sequence:

    • The 48S complex incorporates the initiator tRNA-Met along with eIFs and GTP energies.

    • The complete 80S ribosome is formed when the 60S subunit joins.

Initiation of Translation in Bacteria

  • The process is simpler and more straightforward compared to eukaryotes:

    • No 5′ cap; the ribosome attaches to the Shine–Dalgarno sequence of mRNA.

    • The fMet-tRNA pairs directly with the start codon (AUG).

    • Involves only three initiation factors, contrasting with over 12 in eukaryotes.

    • There is no scanning; the ribosome directly assembles at the start codon.

    • Polycistronic mRNAs allow multiple proteins to be synthesized from a single transcript.

The Role of the Ribosome

  • Ribosomes engage in cyclic GTP-driven mechanical transformations during translation.

  • The information inscribed in the mRNA governs the sequence of aminoacyl-tRNAs accepted by the ribosome.

  • Ribosomes contain three distinct tRNA binding sites:

    • A (aminoacyl): Binds the incoming aminoacyl-tRNA carrying a new amino acid.

    • P (peptidyl): Holds the tRNA associated with the polypeptide.

    • E (exit): Releases the empty tRNA after the amino acid is transferred.

Translation: Elongation

  1. Step 1 – Aminoacyl-tRNA Selection:

    • The second aminoacyl-tRNA binds to the A site, which involves GTPase cooperation.

    • In bacteria: this is facilitated by elongation factor EF-Tu.

    • In eukaryotes: it is accomplished through eEF1A.

    • Hydrolysis of GTP releases EF-Tu (or eEF1A), properly positioning the new aminoacyl-tRNA in the A site for peptide bond formation.

  2. Step 2 – Peptide Bond Formation:

    • Peptide bond formation is catalyzed by peptidyl transferase, an enzymatic activity of the large ribosomal subunit, and does not require external energy.

    • Peptide chain transfers from the P-site tRNA to the A-site tRNA.

    • Outcomes of bond formation:

      • tRNA in the P site becomes uncharged (no amino acid attached).

      • tRNA in the A site now retains the growing peptide chain.

  3. Step 3 – Translocation:

    • An elongation factor binding, supplemented by GTP hydrolysis, enables the ribosome's movement along the mRNA.

    • In prokaryotes: facilitated by EF-G;

    • In eukaryotes: this process is managed by EF-2.

    • The ribosome shifts three nucleotides (one entire codon) toward the 5′ to 3′ direction, leading to:

      • Movement of the dipeptidyl-tRNA from the A site to the P site.

      • Movement of deacylated tRNA from the P site to the E site.

  4. Step 4 – Release of Deacylated tRNA:

    • The empty deacylated tRNA is released from the ribosome, leaving the E site.

    • Important Note:

      • Each elongation cycle consumes at least two GTP molecules.

      • Each tRNA smoothly transitions through three sites: A → P → E, except the initiator tRNA which occupies the P site on initiation.

Termination

  • Translation termination occurs upon the arrival of stop codons, which include UAA, UAG, or UGA.

  • The process necessitates the involvement of release factors, which recognize stop codons and modify the ribosomal peptidyl transferase function:

    • Instead of adding an amino acid, water is introduced to hydrolyze the polypeptide chain terminator.

  • The subsequent step involves the disassembly of the ribosome and release of the mRNA, signifying complete translation.

mRNA Surveillance and Quality Control

  • Mutations can lead to premature stop codons in a gene (termed nonsense mutations) or result from splicing issues.

  • Nonsense-mediated decay (NMD) is a mRNA surveillance system that identifies mRNAs with premature stop codons and triggers their degradation, preventing the production of truncated and non-functioning proteins.

Types of Mutations Affecting Genes

  1. Point mutations: Alter a singular nucleotide (reference chart for classifications).

  2. Frameshift mutations: Involve the insertion or deletion of nucleotides, which shifts the reading frame of the gene.

  3. Splice site mutations: Modify normal mRNA splicing, influencing the addition or removal of exons.

  • Mutation Effects:

    • Synonymous (no change in amino acid).

    • Non-synonymous (changes the amino acid).

    • Stop codon introduction results in:

    • Functional Protein: Proper protein expression.

    • Non-functional Protein: Inhibition or failure in protein function.

Blocking Translation: The Action of Antibiotics

  • Antibiotics may inhibit bacterial growth by:

    • Binding to the bacterial ribosome, obstructing the translation process.

    • Targeting transcription pathways to impede protein synthesis.

  • Binding Sites for Antibiotics on the bacterial ribosome are crucial in understanding their mechanisms of action.

Check Your Understanding

  1. Describe how tRNAs decode codons and how aminoacyl-tRNA synthetases ensure accuracy.

  2. Outline the key steps of translation: initiation, elongation, and termination.

  3. Explain how GTP hydrolysis drives translation and ensures fidelity.

  4. Identify the roles of the A, P, and E sites in peptide chain elongation.

  5. Recognize how mutations and antibiotics can disrupt translation.