Protein Synthesis Flashcards
RNA’s involved in translation
Messenger RNA- carries genetic information from DNA in a linear form via the process of transcription. It is read in sets of 3- nucleotide sequences called CODONS which specify a particular amino acid.
- transfer RNA- key to deciphering codons in mRNA. Each amino acid has its own set of tRNA’s which bind to it. Each tRNA contains a 3-nucleotide sequence- the ANTICODON- which is complementary to the condon in mRNA. Hence the mRNA specifies which tRNA’s and hence which amino acids are next recruited.
- Ribosomal RNA- associated with protein to form the ribosomes, which catalyse the assembly of the amino acids into the polypeptide chain
Cricks adapter hypothesis
- amino acids to not bind directly to mRNA
- crick proposed that an adapter molecule could match the mRNA with a corresponding amino acid
- at one site, the adapter could bind to a specific amino acid
- a separate part of the adapter could recognise the nucleotide sequence in mRNA that encode that amino acid
- adapter molecule is now known as trna
Codon usage
The only possible way that there are enough codons for the 20 different amino acids is if the codon is 3 nucleotides long.
This is why the anticodon on tRNA is three bases in length
The result of this is that the code is highly degenerate meaning that there is more than one triplet that could specify the same amino acid
The wobble base
Where several different codons specify more than one amino acid, variability is most frequently observed at the third base
Special codons
Start codon- AUG
Stop codons/ termination- UAA, UAG, UGA
Reading frames
In a random nucleotide sequence, one in twenty codons is a stop codon
Segments that do not have termination codons for at least 50 codons are called open reading frames
In theory, every mRNA has three potential reading frames.
In most cases, only one is used. Selection of the correct reading frame relies on features upstream of the start codon
How polypeptides are made
-Substrate preparation: amino acid are activated and bound to tRNA. 20 AAs used
-Initiation: assembly synthetic machinery
Ribosome, mRNA, activated amino acid
-Elongation:
Catalyse peptide bond (covalent) between successive amino acids to create a polypeptide
-Termination and release from ribosome
Guessing nucleotide sequence from protein sequence
Can occur, protein must be purified and must know at least 5-10 amino acids. Sequence of N terminal or proteolytic fragments.
Treat protein with protease (trypsin)
Separate peptides on HPLC
Use this sequence obtained to design olgionucleotides
Mutations
- Errors occurring spontaneously during DNA replication lead to permanent changes in sequence
- Somatic mutations are not inherited, only passed on to future generation if they occur in germ- line cells (egg, sperm, precursor cells)
- Can be good (evolution), bad (cancer) or indifferent
- single base change (point mutation) can have no effect if mutated codes for same aa, can result in protein having one different amino acid (harmless or detrimental), truncate the protein if the mutation results in a stop codon.
- mutations at wobble base may not cause change
- insertion of deletions in nucleotide sequence can change that reading frame, resulting in mutant protein. If it is a multiple of three, will still lead to an altered protein, but reading frame will remain intact.
- silent mutation does not affect function
- frame shift mutations involve changing the spacing of the codons- insertion or deletion.
Structural properties of tRNA
Is in a T shape- D arm, TC arm, amino acid arm and anticodon arm
In the attached state, the tRNAs are often referred to as charged.
This process is controlled by enzymes within the cytosol termed aminoacyl tRNA synthetases.
Each aminoacyl tRNA synthetase can recognise a single amino acid, but in some cases, one or more tRNAs.
- amino acids are first activated by the reaction with ATP (to form aminoacyl AMP intermediate) and then joined to the 3’ terminus of the tRNA
- aminoacyl group is transferred to the adenine nucleotide of tRNA- forms aminoacyl-tRNA
Initiating methionine (AUG)
Polypeptide begins with amino-terminal methionine.
All cells have two tRNA’s for this amino acid,none of these is for initiation and the other is for internal methionines.
This initiating 5’ AUG is specifically recognised by tRNAfmet in bacteria, or tRNAimet in eukaryotes. Internal AUG codons are recognised by the other tRNAmet
Initiation: pre-initiation
30s and 50s ribosome subunits are separate until translation begins. Initiation factors (IF1-3) are proteins which control assembly of a functional ribosome. IF3 prevents premature assembly.
mRNA is guided to the correct position by the Shine-Dalgarno sequence. This sequence is a ribosomal binding site in mRNA generally located 8 base pairs upstream of AUG.
exists only in prokaryotes: AGGAGGU. Sequence help recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with a start codon.
Translocation
Involves the ribosome moving a single codon downstream
Driven by elongation factor G that is bound to GTP
The EF-G-GTP factor mimics the shape of the aminoacyl-tRNA-EF-Tu-GTP complex and can enter the A site in a similar manner
Conformational change in the ribosome results in movement along the mRNA
Ribosome moves so the new peptide attached to the tRNA (and mRNA) at the P-site
The used tRNA moves to exit site where it is released
Termination
When the ribosome reaches a stop codon, no corresponding tRNA
Instead another set of accessory proteins called release factors (RF) bind directly to the termination codon
The release factor transfers a H2O to create a carboxy terminus- peptide release
In eukaryotes all three termination codons are recognised by the same RF
Termination leads to the dissociation of the ribosomal complex
Post translational modifications
- removal of signal sequences: polypeptides carry internal messages directing them to a specific cellular location. Once there, these messages can be removed.
- folding: protein must fold from a linear polypeptide into a three dimensional shape.
- addition of groups: sugar groups- important in recognition of events, lipid groups- anchoring of membrane proteins, prosthetic groups- eg heme in haemoglobin
- modification of AA - phosphorylation on certain AAs in enzymes can regulate activity. Formation of disulphides bonds between cysteines can aid in locking in 3D structure
- proteolytic processing- converting an inactive precursor - active form
