protein synthesis

• what are the three properties of the genetic code?

  Universal, degenerate, and non-overlapping.
• what do the stop and stat codons do?

  Stop codons signal the end of protein synthesis, while start codons initiate protein synthesis.
• whats transcription?

  process of making a complementary mRNA strand from DNA (makes an antisense copy)
• why does DNA need to be transcribed to a single strand of mRNA?

  DNA has the genetic code for protein synthesis but it's too large to leave the nucleus as a double strand. But RNA can leave the nucleus through nuclear pores
• what is tRNA?

  another type of RNA used in translation
• whats the structure of tRNA?

  clover shaped, has an amino acid binding site and an anticodon
• where does mRNA go after leaving the nucleus?

  to cytoplasm and binds to ribosome = protein factory
• free mRNA is not a very stable molecule, it won't remain very stable in the cytoplasm for long or it will be degraded so it needs to be pulled in and used
• what is function of the tRNA in translation?

  They can only bind to their specific amino acid (forms covalent bond with AA) and they carry the amino acids to the ribosome and match them with the codons on the mRNA
• whats an anticodon and codon?

  Anticodon: A sequence of three nucleotides on a tRNA molecule that is complementary to a specific codon on mRNA Codon: A sequence of three nucleotides on mRNA that codes for a specific amino acid or serves as a start or stop signal during protein synthesis
• an anticodon and codon are complementary
• the ribosome provides stability and shelter + allowing the mRNA and tRNA to meet
• what is translation?

  the decoding of an mRNA message into a polypeptide chain
• how are amino acids attached to their cognate tRNA?

  through a two step process called aminoacylation

  1. Activation of amino acid
  2. Transfer to tRNA
• what occurs in the activation of an amino acid?

  The amino acid is activated by the addition of ATP to form aminoacyl-AMP
• what occurs after the activation of an amino acid?

  the activated amino acid (aminoacyl-AMP) binds to the 3' end of its tRNA which is catalysed by a specific tRNA synthetase (e.g for the AA lysine the enzyme is lysine/tRNA synthetase)
• in RNA - A binds to U and C to G
• what are the three main stages of translation?

  Initiation, elongation cycle, termination.
• describe initiation stage in translation?

  • small ribosomal subunit that has initiation factors and base pairings binds to the mRNA near its 5' end
  • the start codon (AUG) of mRNA is positioned in the ribosomal P site and the initiator tRNA (formyl-methionyl tRNA) binds to it
  • once start codon is recognised, the large subunit associates with the small subunit
  • GTP (guanosine triphosphate) is hydrolysed and initiation factors leave the ribosome. The energy allows all of the connection movement to start
• what are initiation factors?

  proteins that help in the assembly of the translation machinery
• what are the sites where tRNA can bind called?

  A, P and E sites

  • only two of these are normally occupied at one time - one that's doing something, one that's about to be connected and a space
• what are the A, P and E sites function?

  A site: Aminoacyl site, where the incoming aminoacyl-tRNA binds P site: Peptidyl site (bond formation)

  E site: Exit site, where the deacylated tRNA exits after its amino acid has been transferred to the growing polypeptide chain during protein synthesis
• what are the steps in elongation of translation?

  • aminoacyl-tRNA binding
  • peptide bond formation
  • translocation
• During elongation in translation, the aminoacyl tRNA carrying the next amino acid (determined by the codon in the A site) binds to the A site, a process facilitated by an elongation factor.
• elongations step 2 - The enzyme peptide transferase catalyses the formation of a peptide bond between the amino group of the new amino acid in the A site and the carboxyl group of the growing polypeptide chain in the P site, resulting in the transfer of the growing polypeptide chain in the P site to the A site.
• elongation 3) The ribosome moves 1 codon towards the 3' end of the mRNA so that the GPC moves from A to P site, a process facilitated by EFG, which means the unloaded tRNA moves to the E site and A site is empty again.
• describe the termination stage in translation?

  • synthesis of the chain continues until a stop codon is reached (UAA, UGA or UAG)
  • a special release factor binds in the A site - RFs do not have an amino acid on and breaks covalent bond of the last tRNA binder to its amino acid to release the polypeptide chain
  • polypeptide in the P site is then hydrolysed from its tRNA and leaves via the exit tunnel, the tRNA the exits the ribosome
  • the release is catalysed by 2x hydrolysis of GTP
  • ribosomal subunits dissociate ready to be used again
• the exit tunnel - has a specific shape in the ribosome
• many antibiotics work by binding to the bacterial ribosome and disrupting their function - due to bacteria having different structural ribosome than us, antibiotics can be designed that affect/bind to their ribosomes and not our own
• name examples of antibiotics that affect protein synthesis?

  • inhibition of initiation - Streptomycin
  • prevention of large subunit binding - Linezolid
  • prevention of tRNA binding to the A site - Tetracycline
  • inhibition of peptide bond formation (P site function affected) - Chloramphenical
  • blocking the exit tunnel of the ribosome - erythromycin
• how does tetracycline affect protein synthesis?

  Prevents tRNA (with amino acid) from binding to the A site during the elongation cycle - and so prevents the addition of new amino acids to the growing polypeptide chain

  • common use in treatment = moderately severe acne + skin infections
• how does chloramphenicol affect protein synthesis?

  Inhibits the peptide bond formation - interferes with the peptidyl transferase - results in incomplete proteins

  • effective against a whole range of bacteria. however, because of resistance, it's no longer the first line agent for infection in developed nations, except for topical treatment of bacterial conjunctivitis
• how does erythromycin affect protein synthesis?

  Blocks exit tunnel
• mutation of just a single base in the tRNA or a single amino acid in the ribosomal protein can cause antibiotic resistance

  • if a few bacteria survive, they can start to change and mutate in response to the new environment and so the antibiotic can no longer bind to them
  • using antibiotics all the time increases the selection pressure and opportunity for resistance
• mRNA can be chopped = splicing - it makes it slightly different so have slightly different functions

  • so there can be 100,000 different mRNA even through we have around 30,000 genes - slight variation causes this
• whats post-translational modifications?

  Once mRNA has been translated into the sequence of amino acids it gets dropped from the ribosome and how that folds and how additives are added can make a huge difference to how they function and what they do
• a single gene can give a rise to multiple RNA transcripts and each transcript can be modified to give multiple versions of a single protein
• PTM is of fundamental importance for biological activity of proteins - its plays a key role in the activity, localisation and interactions of proteins with other cellular models
• • modifications have significant effects on protein structure and function and are of concern for proteins of therapeutic or diagnostic use
  • the proteome is dynamic and changes in response to stimuli, PTMs are important in regulating cellular activity
• what are common types of PTMs?

  • phosphorylation
  • glycosylation
  • lipidation
  • acetylation
  • ubiquitination
  • methylation