Protein Encoding and Translation

• The lecture focuses on encoding proteins, or translation.

Recommended Biochemistry Textbook

• Biochemistry by T.A. Brown can be used as a resource.

Central Dogma of Molecular Biology

• DNA undergoes replication to produce more DNA.
• DNA undergoes transcription to produce RNA.
• RNA undergoes translation to produce protein, which leads to cellular function.
• Transcription is the enzymatic synthesis of RNA from a DNA template and forms the first step in gene expression
• Translation is the enzymatic synthesis of protein from an transcribed gene sequence into a functional RNA molecule mRNA

Protein Structure

• Primary structure is the amino acid sequence in a polypeptide chain.
• Secondary structure refers to local folded structures formed by interactions between backbone atoms.
• Tertiary structure is the overall 3D structure of a polypeptide, resulting from interactions between R groups of amino acids.
• Quaternary structure arises when multiple polypeptide chains (subunits) combine.

Amino Acids

• Amino acids are the building blocks of proteins and form peptide bonds.

Translation

• Translation is the process where ribosomes read the transcribed mRNA to generate a protein.
• In prokaryotes, translation is co-transcriptional.
• In eukaryotes, translation occurs after transcription, though some elements may be co-transcriptional.
• Translation occurs in the cytoplasm.

The Ribosome

• Responsible for reading the mRNA sequence and assembling the protein based on that message.
• It has a diameter of approximately 10 nm.
• Ribosomes consist of about 65% ribosomal RNA (rRNA) and 35% ribosomal proteins.
• Ribosome units are characterized by their sedimentation rate in Svedberg units (S).
• Under optimal conditions, rRNA and protein mixtures self-assemble into a ribosome.
• Ribosomes' RNAs have both structural and catalytic activity.
• The small ribosomal subunit contains the decoding center required for reading mRNA.
• The large ribosomal subunit contains the peptidyl transferase center, which has catalytic RNA.
• Multiple ribosomes loaded onto a single mRNA strand are called a polyribosome.
• Transfer RNAs (tRNAs) occupy the A, P, and E sites and carry amino acids.
• The A-site is the acceptor site where amino-acyl tRNA lands.
• The P-site is occupied by peptidyl-tRNA, where the last amino acid was added.
• The E-site is the exit site where tRNA leaves the ribosome after transferring its amino acid.
• The A, P, and E sites lie in the large subunit but are completed when the small unit is present.

Transfer RNAs (tRNAs)

• Adapter molecules that deliver amino acids to the ribosome.
• The primary structure of a tRNA is between 60 and 95 nt long (most commonly 75 nt) that contains many (>20%) post-transcriptionally modified bases
• All tRNAs have the same basic secondary 'clover-leaf' structure
• There are significant levels of intra-tRNA hydrogen bonding sites

Reading the Genetic Code

• mRNA sequence carries its message in 3-letter "words".
• The genetic code is essentially a continuous run of 3 nucleotide triplets known as codons.
• Translation is the process whereby the codons are read and the information used to insert an amino-acid into the growing polypeptide
• Virtually every amino-acid is encoded by more than one codon - a redundancy is built-in to the code

Deciphering the Genetic Code: Nirenberg Experiment

• Marshall Nirenberg and his team synthesized mRNA with repeating nucleotides for their experiments.
• They added these synthetic mRNAs to E. coli cell lysate.
• The resulting proteins were isolated to see what amino acids were incorporated.
• 5′-UUUUUUUU-3′-OH gave poly-Phenylalanine.
• 5′-AAAAAAAA-3′-OH gave poly-Lysine
• 5′-CCCCCCCC-3′-OH gave poly-Proline
• Repeating 5′-AUAUAU.....3′-OH resulted in a protein containing alternating isoleucine and tyrosine.

Translation in Prokaryotes

• Components needed for translation include mRNA, tRNA, ribosomes, GTP, initiation factors, and elongation factors.
• Translation occurs in 3 stages: initiation, elongation, and termination.
• Movement of the ribosome along the mRNA is called translocation.
• Purpose of the initiation step is to assemble the translation “machinery" at the translation start site
• Initiator tRNA enters the P-site, while all subsequent tRNAs enter the A-site.
• In addition, 3 initiation factors and a molecule of GTP are required
• The elongation step involves aminoacyl-tRNA delivery, peptide bond formation, and translocation.
• After initiation, the P-site is occupied, and the A-site is empty.
• Three elongation factors are recruited, all can bind GDP or GTP.
• The ribosome complex maintains a 6 bp contact with the mRNA, which stops frame-shifting.
• Elongation continues until a termination codon is read in the A-site.
• Termination occurs when there are no tRNAs that recognize a STOP codon
• Protein release factors interact with these codons and bring about the release of the polypeptide chain
• Release Factor 1 (RF1) recognises UAA and UAG
• RF 2 recognises UAA and UGA
  • RF3 helps RF1 and RF2 to carryout their role
• Release factor causes the peptidyl transferase to transfer the polypeptide to water rather than to the next tRNA – so the protein is then released
• EF-G and a RF are required for the disciation of the ribosome complex from the mRNA, and removal of the uncharged tRNA from the P-site

Co-translational Control: The Tryptophan Operon

• Transcription and translation occur simultaneously in bacterial cells.
• As mRNA is generated, it is also being read by a ribosome (protein production)
• It uses this co-translation as a control point for gene expression regulation.
• The trp operon, found in E. coli, is a group of genes that encode biosynthetic enzymes for tryptophan, a rare amino acid.
• The trp operon is expressed (turned "on") when tryptophan levels are low and repressed (turned "off") when they are high.
• It is regulated by the trp repressor, which blocks expression when bound to tryptophan.
• Biosynthesis is also regulated by attenuation, a process based on coupling transcription and translation.
• Attenuation functions as a control mechanism when tryptophan levels are high, stopping ribosome progression along the mRNA.
• Control sequence is used to respond to tryptophan levels
• It lies between the operator and the first gene if the operon and is called the leader sequence
• The leader sequence encodes a short polypeptide and also contains an attenuator sequence.

Interfering with Protein Translation

• Chloramphenicol binds to 50S & inhibits formation of peptide bond
• Macrolides, Clindamycin & Streptogramins block the polypeptide exit tunnel on 50S & prevent peptide chain elongation
• Linezolid binds to 50S & prevents formation of the 50/30S ribosomal complex
• Aminoglycosides bind to 30S & causes mRNA codon to be misread; interfere with the initiation complex of 30S & 50S with mRNA
• Tetracyclines bind to 30S & interfere with binding of tRNA to ribosomal complex

Protein Physicochemical Properties

• Contains carbon, hydrogen, oxygen, nitrogen
• Will have a defined structure/shape
• Will have a defined mass
• Will absorb light in the UV range
• Will contain both charged and uncharged amino acids
• Will contain hydrophilic and hydrophobic amino acids

Summary

• How the mRNA sequence is turned into protein
• How the genetic code was elucidated experimentally
• How transcription and translation can be combined for gene expression control
• How we can interfere with the translation process