Translation Flashcards

The Genetic Code

• The amino acid sequence of a protein is determined by the sequence of bases on the DNA strand.

• The coding problem is how four bases code for 20 amino acids.

  • Pairs: 4^2 = 16 permutations - not enough.

  • Triplets: 4^3 = 64 permutations - more than enough.

  • Therefore, the code is degenerate.

• Amino acids are encoded by triplets of bases (codons).

  • A sequence of 3 DNA/RNA bases encodes for one amino acid.

• Determining which bases encode which amino acid:

  1. Make synthetic mRNA.

  2. Find out which amino acids are incorporated into protein in a cell-free system (Matthaei and Nirenberg, c. 1962).

Experimental Elucidation of the Genetic Code

• Cell-free system (ribosomes, amino acids) is used.

• Only one amino acid is radio-labeled per tube (e.g., serine, leucine).

• The mixture is poured onto filter paper to wash off free amino acids and mRNA, leaving ribosomes stuck to the filter paper.

• The filter paper is scanned for a radioactive signal.

• If the tube containing labeled phenylalanine has a high signal (above background), it indicates that UUU encodes phenylalanine, as other amino acids do not show a signal.

• The genetic code is read in triplets.

• The code is degenerate, meaning that 64 codons code for 20 amino acids.

• One start codon (AUG) signals the start of translation (all proteins start with methionine (Met)).

• Three stop codons signal the termination of translation/end of protein.

• Reading frames are determined by where you begin to read the code, which determines which triplets are produced. Each possible grouping of triplets is a reading frame.

Deciphering Challenges and Solutions

• Initially, Nirenberg’s lab could not control the order in which nucleotides were polymerized.

  • For example, putting A, G, and C in a tube could yield AAA, AAC, AAG, ACA, AGA, ACG, etc.

• Har Gobind Khorana worked out how to synthesize RNA with a specific sequence, which made assigning codons to amino acids easier.

• Khorana shared the 1968 Nobel Prize with Nirenberg and Robert W. Holley (who determined the structure of alanine tRNA) for deciphering the genetic code.

Summary of Part A

• The genetic code was deciphered using synthetic mRNA and evaluating which amino acids were incorporated.

• Amino acids are encoded by triplets of nucleotide bases (codons).

• The code is degenerate – most amino acids are coded for by more than one codon.

• One start codon (AUG) and three stop codons.

• Where you begin to read the code will determine which triplets are produced; each possible grouping of triplets is called a ‘reading frame’.

Mechanism of Translation

• The mechanism of translation involves:

  • mRNA

  • Ribosomes

  • tRNA

• mRNA encodes proteins.

• rRNA is a structural and enzymatic component of the ribosome.

• tRNA delivers amino acids to the ribosome.

• Transcription involves complementarity.

• Translation involves amino acids that cannot pair directly with RNA codons, thus requiring an adaptor.

• Transfer RNA (tRNA) is the adaptor molecule that links an mRNA codon with a specific amino acid.

Transfer RNA (tRNA) Molecule

• tRNA structure:

  • 75-90 nucleotides.

  • Extensive internal base pairing.

  • Clover leaf structure.

  • Contains unusual bases.

  • CCA-OH sequence at the 3’ end.

  • Anticodon on the central loop.

• The anticodon is a triplet of bases that are complementary to the codon.

• These bases are unpaired and available for hydrogen bonding.

• There are 61 amino acid codons but fewer than 61 tRNA molecules, therefore some tRNAs must recognize more than one codon.

• ‘Wobble pairing’ allows this to happen, where the third base can show ‘wobble’.

• Only tryptophan and methionine are encoded by a single codon.

• Often the first 2 letters in the codon are the same, and the third base can vary.

• The third position is less critical and can follow non-Watson-Crick base pairing between mRNA and tRNA.

• This allows a single tRNA species to recognize more than one codon without altering the amino acid sequence of a protein.

• A ‘charged’ tRNA has an amino acid attached at the 3’ end via an ester linkage.

• Each tRNA can accept only the single amino acid that is appropriate for its anticodon sequence.

• Specific aminoacyl-tRNA-synthetases ‘load’ tRNA molecules with amino acids.

Charging tRNAs

• Energy for the addition of the amino acid to tRNA comes from the hydrolysis of ATP: amino acid + tRNA + ATP \rightarrow aminoacyl-tRNA + PPi + AMP

• There are twenty different types of tRNA, classed by their amino acid (e.g., tRNAphe, tRNAleu, tRNAtyr, etc.).

• When they are charged, they are sometimes called ‘activated amino acids’ and named differently (e.g., phe-tRNAphe, leu-tRNAleu, tyr-tRNAtyr, etc.).

Ribosome Structure

• The E. coli ribosome is a 70S complex.

  • Large subunit (50S): 23S rRNA + 5S rRNA + 34 proteins

  • Small subunit (30S): 16S rRNA + 21 proteins

• Ribosomes have three tRNA sites: E-site, P-site, and A-site.

• In eukaryotes, polyribosomes are free in the cytoplasm or can be bound to rough endoplasmic reticulum (RER).

Summary of Part B

• Translation requires RNA and ribosomes (and amino acids).

• Transfer RNAs (tRNAs) function as adaptors, linking mRNA codons with specific amino acids.

• Amino acids are attached via an ester linkage to the 3’ end of the tRNA. The central loop of the tRNA contains an anticodon, which can pair with an mRNA codon.

• Wobble pairing allows a single tRNA anticodon to recognize more than one codon.

• tRNAs are charged with the correct amino acids by specific aminoacyl-tRNA-synthetases.

• Ribosomes have a small and large subunit, and three tRNA sites (E, P, and A).

Mechanism of Translation: Initiation, Elongation, and Termination

• Like transcription, the mechanism of translation has 3 parts:

  • Initiation

  • Elongation

  • Termination

Prokaryotic Translation: Initiation

• Initiation factors (IFs) IF1 and IF3 bind the 30S subunit.

• This complex binds mRNA.

• fMet-tRNAfMet in complex with IF2-GTP enters the P site.

• 16S rRNA binds to the Shine-Dalgarno sequence in the mRNA to line up fMet-tRNAfMet with the AUG start codon (Shine-Dalgarno sequence is in the 5’ UTR of the mRNA).

• The large 50S subunit binds.

• Accompanied by hydrolysis of GTP.

• GDP + Pi, and IFs (1, 2, 3) are released.

Prokaryotic Translation: Elongation

• The next aminoacyl tRNA binds to elongation factor EF-Tu-GTP and enters the A site in the ribosome.

• If the anticodon of the incoming tRNA is complementary to the codon, then hydrolysis of GTP takes place, and EF-Tu-GDP + Pi are released.

• The protein is synthesized by ‘lifting’ the incomplete polypeptide and placing the incoming (‘new’) amino acid underneath.

• The free –NH2 of the incoming amino acid attacks the carbonyl carbon of the previous amino acid to form the peptide bond.

• Translocation of the ribosome occurs with hydrolysis of the GTP bound to EF-G. The A site is now free again.

• The discharged tRNA is released from the E site.

Prokaryotic Translation: Termination

• A stop codon (UAA, UAG, or UGA) on mRNA is presented in the A site.

• Release factor (RF1 or RF2) mimics the shape of a tRNA.

• The release factor enters the A site with an H_2O molecule.

• The peptide is hydrolyzed from the final tRNA using an H_2O molecule.

• The ribosome disassembles, which requires a ribosomal recycling factor and IF3.

Prokaryotes vs. Eukaryotes

Prokaryotes

• Transcription occurs in the cytoplasm.

• Polycistronic mRNA that encodes more than one protein; the mRNA is unmodified.

• Translation also occurs in the cytoplasm, so mRNA can be translated as soon as it is synthesized (or while being synthesized).

• 30S + 50S = 70S ribosome.

• Polyribosomes are free in the cytoplasm.

• The ribosome is positioned at the start codon by the interaction of the 16S rRNA with the Shine-Dalgarno sequence.

• The initiator tRNA is fmet-tRNAfmet.

Eukaryotes

• Transcription occurs in the nucleus.

• hnRNA/pre-RNA is modified by capping, poly-A tail, and splicing before export from the nucleus.

• The mRNA is monocistronic, encoding for one protein only.

• Translation occurs in the cytoplasm, so mRNA cannot be translated as soon as it is synthesized because it first has to be processed and exported from the nucleus.

• 40S + 60S = 80S ribosome.

• Polyribosomes are free in the cytoplasm or can be bound to rough endoplasmic reticulum (RER).

• The 5’ cap interacts with the ribosome – it is not clear how the start codon is found.

• The initiator tRNA is met-tRNAmet.

Summary of Part C

• Like transcription, the mechanism of translation has three parts – initiation, elongation, and termination.

• Initiation requires initiation factors (IFs), fMet-tRNAfMet in complex with IF2-GTP, and hydrolysis of GTP accompanied by the binding of the large 50S subunit.

• 16S rRNA binds the Shine-Dalgarno sequence in the mRNA to line up fMet-tRNAfMet with the AUG start codon.

• During elongation, the nascent polypeptide is synthesized in the P site.

• Translation is terminated by a release factor that mimics the shape of a tRNA.

• There are key differences between prokaryotic and eukaryotic translation.