Protein Synthesis: Translation

Proteins – Introduction

  • Translation is the process by which the information in the nucleotide sequence of mRNA is converted into an amino acid sequence.

  • Proteins are active participants in cell structure and function.

  • The start codon sets the reading frame for all remaining codons.

  • Anticodons are complementary to codons.

Genetic Code

  • The triplet codon represents each amino acid.

  • There are 20 amino acids encoded by 4 nucleotides.

    • 1 nucleotide/amino acid = 4^1 = 4 combinations

    • 2 nucleotides/amino acid = 4^2 = 16 combinations

    • 3 nucleotides/amino acid = 4^3 = 64 combinations

  • There must be at least triplet combinations that code for amino acids.

Cracking the Code

  • Several technological breakthroughs in the 1950s and 1960s helped crack the genetic code:

    • In vitro translation of synthetic mRNAs.

    • Preparation of cellular extracts that allowed translation in a test tube.

    • Developed techniques to synthesize artificial RNAs with known nucleotide sequences.

Using Synthetic mRNAs and In Vitro Translation

  • 1961 – Marshall Nirenberg and Heinrich Matthaei added artificial mRNAs to cell-free translation systems.

Nobel Prizes

  • Severo Ochoa and Arthur Kornberg (1959): For their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid.

  • Robert W. Holley, H. Gobind Khorana, and Marshall W. Nirenberg (1968): For their interpretation of the genetic code and its function in protein synthesis.

Triplet Binding Assay

  • Nirenberg and Leder (1965) resolved ambiguities in the genetic code using in vitro translation with trinucleotide synthetic mRNAs and tRNAs charged with a radioactive amino acid.

Genetic Code Characteristics

  • Triplet codons of nucleotides represent individual amino acids.

  • 61 codons represent the 20 amino acids.

  • 3 codons signify stop.

  • The genetic code is almost universal; virtually all cells alive now use the same basic genetic code.

    • In vitro translational systems from one organism can use mRNA from another organism to generate protein.

    • Comparisons of DNA and protein sequence reveal perfect correspondence between codons and amino acids among all organisms.

  • The genetic code must have evolved early in the history of life; exceptions are found in ciliates and mitochondria.

Structure of tRNAs and Ribosomes

  • Transfer RNAs (tRNAs) mediate translation of mRNA codons to amino acids.

  • Translation takes place on ribosomes that coordinate the movement of tRNAs carrying specific amino acids.

  • tRNAs are short single-stranded RNAs of 74 – 95 nt.

    • Each tRNA has an anticodon that is complementary to an mRNA codon.

    • A specific tRNA is covalently coupled to a specific amino acid (charged tRNA).

    • Base pairing between an mRNA codon and an anticodon of a charged tRNA directs amino acid incorporation into a growing polypeptide.

tRNA Functions

  1. It brings amino acids to the site of protein synthesis (i.e., Ribosomes).

  2. It reads the codon on mRNA to incorporate the correct amino acid.

Recognition Between tRNA and mRNA

  • During mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary codon in mRNA.

  • tRNAs are named according to the amino acid they bear.

  • The anticodon is anti-parallel to the codon.

Modified Bases in tRNA

  • Some tRNAs contain modified bases such as Ribothymidine (T), Dihydrouridine (UH2), Pseudouridine (\Psi), 4-thiouridine (S4U), Inosine (I), etc.

Three Levels of tRNA Structure

  • Nucleotide sequence is the primary structure.

  • Secondary structure (cloverleaf shape) is formed because of short complementary sequences within the tRNA.

  • Tertiary structure (L shape) is formed by 3-dimensional folding.

Aminoacyl-tRNA Synthetases

  • Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to specific tRNAs.

  • Each aminoacyl-tRNA synthetase recognizes a specific amino acid and the structural features of its corresponding tRNA.

  • There are 20 types, one for each amino acid.

Base Pairing

  • Base pairing between the mRNA codon and tRNA anticodon determines which amino acid is added.

Wobble

  • Some tRNAs recognize more than one codon for the amino acid they carry.

  • Wobble Rules: 5' end of anticodon can pair with 3' end of codon.

Ribosomes

  • Translation occurs on the surface of a large macromolecular complex termed the ribosome.

  • Ribosomes are complexes of rRNAs and Protein.

  • Each ribosome has two subunits (one large and one small).

Ribosome Functions

  • They recognize mRNA and find the initiation codon.

  • Stabilize the interaction between mRNA and tRNA.

  • They contain the enzyme to create the peptide bond.

  • Ensure that codons are read one after another in sequence.

  • Help terminate protein synthesis.

Ribosome Composition

  • Prokaryotic:

    • 70S ribosome

    • 50S subunit (23S rRNA of 3000 nucleotides, 5S rRNA of 120 nucleotides, 31 proteins)

    • 30S subunit (16S rRNA of 1700 nucleotides, 21 proteins)

  • Eukaryotic:

    • 80S ribosome

    • 60S subunit (28S rRNA of 5000 nucleotides, 5.8S rRNA of 160 nucleotides, 5S rRNA of 120 nucleotides, ~45 proteins)

    • 40S subunit (18S rRNA of 2000 nucleotides, 33 proteins)

Ribosome Subunits

  • Small subunit binds to mRNA.

  • Large subunit has peptidyl transferase activity.

  • Three distinct tRNA binding areas – E, P, and A sites.

Nobel Prize

  • Ada E. Yonath, Venkataraman Ramakrishnan, and Thomas Steitz (2009) for studies of the structure and function of the ribosome.

Steps in Protein Synthesis

  • Initiation: All reactions that precede the formation of the first peptide bond between the first two amino acids.

  • Elongation: All reactions from the formation of the first peptide bond to the addition of the last amino acid.

  • Termination: All reactions that are involved in stopping translation and release of the completed polypeptide chain.

Mechanism of Translation

  • Initiation stage: start codon is AUG at the 5’ end of mRNA

    • In bacteria, the initiator tRNA has formylated methionine (fMet).

  • Elongation stage: amino acids are added to the growing polypeptide

    • Ribosomes move in the 5’-to-3’ direction along the mRNA.

    • 2-15 amino acids are added to the C-terminus per second.

  • Termination stage: polypeptide synthesis stops at the 3' end of the reading frame

    • Recognition of nonsense codons.

    • Polypeptide synthesis halted by release factors.

    • Release of ribosomes, polypeptide, and mRNA.

Translation Initiation in Prokaryotes

  • Ribosome binding site consists of a Shine-Dalgarno sequence and an AUG.

  • Three sequential steps: small ribosomal subunit binds first, fMet-tRNA positioned in the P site, large subunit binds.

  • Initiation factors (IF1, IF2, IF3) play a transient role.

Shine-Dalgarno Sequence

  • The binding of mRNA to the 30S subunit is facilitated by a ribosomal-binding site or Shine-Dalgarno sequence, which is complementary to a sequence in the 16S rRNA.

Translation Initiation in Eukaryotes

  • Small ribosomal subunit binds to the 5' cap, then scans the mRNA for the first AUG codon.

  • Initiator tRNA carries Met (not fMet).

  • Eukaryotic Initiation factors - eIF1, eIF2, eIF3, eIF4A, eIF4B, eIF4E, eIF4F, eIF4G, eIF4H, eIF5, eIF6.

  • No Shine-Dalgarno Sequence.

Translation Elongation

  • Addition of amino acids to the C-terminus of the polypeptide.

  • Charged tRNAs ushered into the A site by elongation factors.

    • Prokaryotic Elongation factors - EF-Tu, EF-Ts (brings charged tRNA to A-site), EF-G (moves ribosome by 3 bases).

    • Eukaryotic Elongation factors - eEF1 (brings charged tRNA to A-site), eEF2 (moves ribosome by 3 bases).

Polyribosomes

  • Polyribosomes consist of several ribosomes translating the same mRNA, allowing simultaneous synthesis of many copies of a polypeptide from a single mRNA.

Translation Termination

  • No normal tRNAs carry anticodons for the stop codons.

  • Release factors bind to the stop codons, leading to the release of ribosomal subunits, mRNA, and polypeptide.

    • Prokaryotes: RF1 = UAA, UAG; RF2 = UAA, UGA

    • Eukaryotes: eRF = UAA, UAG, UGA

Antibiotics

  • Most antibiotics are protein synthesis inhibitors, specifically inhibiting bacterial protein synthesis.

    • Streptomycin: Inhibits initiation of protein synthesis; binds to the 30S subunit.

    • Tetracycline: Inhibits elongation; prevents tRNAs from binding to the “A” site.

    • Chloramphenicol: Inhibits elongation; inhibits peptidyl transferase.

Posttranslational Processing

  • Posttranslational processing can modify polypeptide structure through:

    • Cleavage to remove an amino acid.

    • Cleavage to split a polyprotein.

    • Addition of a chemical constituent to modify a protein.

Types of Mutations

  • Silent mutation

  • Missense mutation

  • Nonsense mutation

  • Frameshift mutation

Missense Mutations

  • Conservative: Chemical properties of the mutant amino acid are similar to the original amino acid (e.g., aspartic acid [(-) charged] à glutamic acid [(-) charged]).

  • Nonconservative: Chemical properties of the mutant amino acid are different from the original amino acid (e.g., aspartic acid [(-) charged] à alanine [uncharged]).

Other Mutations

  • Nonsense mutations change a codon that encodes an amino acid to a stop codon (UGA, UAG, or UAA).

  • Frameshift mutations result from the insertion or deletion of nucleotides within the coding region (no frameshift if multiples of three are inserted or deleted).

  • Silent mutations do not alter the amino acid sequence due to the degenerate genetic code, where most amino acids have >1 codon.

Prokaryotes vs. Eukaryotes

  • Prokaryotes:

    • No nucleus; transcription and translation take place in the same cellular compartments, and translation is often coupled to transcription.

    • Genes are not divided into exons and introns.

    • One RNA polymerase consisting of five subunits.

    • DNA sequences needed for transcription initiation are located close to the promoter.

    • Promoters are not wound up in chromatin.

    • Primary transcripts are the actual mRNAs; they have a triphosphate start at the 5′ end and no tail at the 3′ end.

    • Unique initiator tRNA carries formylmethionine.

    • mRNAs have multiple ribosome binding sites (RBSs) and can thus direct the synthesis of several different polypeptides.

    • Small ribosomal subunit immediately binds to the mRNA’s ribosome binding site.

  • Eukaryotes:

    • Nucleus separated from the cytoplasm by a nuclear membrane. Transcription takes place in the nucleus, while translation occurs in the cytoplasm. Direct coupling of transcription and translation is not possible.

    • The DNA of a gene consists of exons separated by introns; the exons are defined by posttranscriptional splicing, which deletes the introns.

    • Several kinds of RNA polymerase, each containing 10 or more subunits; different polymerases transcribe different genes.

    • Enhancer sequences far from the promoter are often needed for transcription initiation.

    • Transcription initiation requires promoters to be cleared of chromatin to allow access to RNA polymerase.

    • Primary transcripts undergo processing to produce mature mRNAs that have a methylated cap at the 5′ end and a poly-A tail at the 3′ end.

    • Initiator tRNA carries methionine.

    • mRNAs have only one start site and can thus direct the synthesis of only one kind of polypeptide.

    • Small ribosomal subunit binds first to the methylated cap at the 5′ end of the mature mRNA and then scans the mRNA to find the ribosome binding site.