S1: Protein Synthesis and Mechanism of Action of Antibiotics Flashcards
Compare the abundance of the different RNA
rRNA = 80-85% tRNA= 10-15% mRNA= 2-5%
How are RNAs size measured?
In the past, their sized was described by their sedimentation coefficient.
Nowadays, they are measured using their nucleotides
What is the coding region in mRNA?
The central portion of mRNA that is translated into protein.
What are the mRNA sequences either side of the coding region called?
Untranslated regions (UTRs) There is a 5'-UTR and 3'-UTR
They do not code for protein
What does the 5’ UTR do?
It determines the rate at which the protein is synthesised
What does the 3’ UTR do?
It affects the stability of RNA (how long it stays in cell before degrading)
Why are the UTR important?
Together, the UTRs determine how much protein is made, the speed at which it is synthesised and the longevity of the message
How is the formation of 5’ cap and 3’polyadenylated tail different from the coding region and UTR?
They are not coded for by genes
They are formed through enzymatic processes on the primary transcript
What is the initiating codon (always) in the coding region of mRNA?
AUG (methionine)
How does tRNA attach to mRNA?
The triplet codes on mRNA (codon) are complementary to the anticodon on tRNA and they base pair. The amino acid is attached to the 3’ end of tRNA
What 3 bases does tRNA have at the 3’ end which has amino acid attached?
CCA
How many possible codons are there?
Triplet codons= 3 bases 4 possible bases 4x4x4 combinations= 64 possible codons
Why are there only 20 amino acids but 64 codons?
This represents a type of redundancy with more than one codon for the same amino acid (degenerate code)
- not all codons code for amino acids (e.g. stop codons)
Why are some codons degenerate?
It protects against the deleterious effects of mutations (increases the result of silent mutations)
What are STOP codons?
They tell the ribosome to stop translating
How does DNA support evolution?
The genetic code is used by the majority of biological organisms (with notable variations)
What are the 4 main steps in the synthesis of proteins?
- Charging tRNAs with amino acids
- Initiation of polypeptide synthesis
- Elongation of polypeptide (addition of amino acid, one at a time)
- Termination of polypeptide synthesis (release of polypeptide)
Why is the correct attachment of the amino acid to tRNA important?
This is because it’s the tRNA that recognises the codon, and if the wrong amino acid attaches then the wrong amino acid will be delievered to the ribosome.
This step is therefore crucial in getting the right protein
What enzyme attaches the amino acid to tRNA?
Aminoacyl tRNA synthase
How does Aminoacyl tRNA synthase attach amino acid to tRNA?
It uses ATP to attach the amino acid to 3’ end of the tRNA.
1 ATP per amino acid
What is the specificity of aminoacyl tRNA synthase referred to as?
Second genetic code
What is the shape of tRNA described as?
Clover leaf structure
Describe the structure of 80s eukaryotic ribosome
2 subunits:
- Large (60s)
- Small (40s)
They assemble together on the initiation of protein synthesis.
There are three parts to the subunit:
- Exit (where the consumes tRNA (lost AA) leaves)
- Peptide (as polypeptide is synthesised, it gets extruded out of the peptide groove in the ribosome)
- Aminoacyl groove (where next amino acid comes in and sits)
(order EPA)
What is the 80s eukaryotic ribosome’s main function?
Functional machine in translation
How does the ribosome assemble on the mRNA?
This involves initiation factors which recognise bits of the mRNA message.
As the mRNA is translated, it usually has a loop structure
How do initiation factors bind to ‘special’ tRNA?
The initiation factors assemble on the small subunit(of the ribosome) and have GTP bound.
What is ‘special’ tRNA?
tRNA which has methionine bound to it.t
What are the two types of ‘special’ methionine tRNA?
- One which goes to AUG codon to initiate translation
2. One is involved in adding methionines to the polypeptide
Where does the initiation tRNA (special) bind to?
TheinitiationtRNAis the only one that can bind to thepeptidebindingsiteon the small ribosomal subunit.
Where do normal tRNAs bind to on the ribosome?
THe aminoacyl site
Where does the small subunit of the ribosome bind to on mRNA?
5’ cap end of mRNA
Is the initiation codon at the 5’ end of mRNA?
No
The actual initiation codon may be further down so the ribosome needs to move along the message until it finds the initiating AUG. This uses ATP.
What happens once ribosome has found initiating codon?
The large subunit is added on which results in the hydrolysis of GTP (on initiation factor). This forms the initiation complex.
What is the initiation complex?
The assembly of the ribosomal subunits and initiator tRNA (met-tRNA) at the start codon on the mRNA.
Explain elongation
The next amino acid is determined by the codon on the mRNA so the aminoacyl tRNA sits in the A site. This involves an elongation factor (EF 1 ) and hydrolysis of GTP as it is energy dependent.
The peptide is added onto the next amino acid using the enzyme peptidyl transferase.
The tRNA becomes distorted due to the first tRNA sat in the P site without being bound to the peptide chain while the second tRNA in the A site is now being bound - peptide still sits in P groove
Translocation is controlled by the enzyme translocase where the ribosome moves one codon along the mRNA and this puts the new tRNA into the P site and the old tRNA into the E site. This involves another elongation factor (EF2) and GTP hydrolysis
Discharge of the used tRNA occurs
What does the enzyme peptidytransferase do?
This enzyme transfers thegrowingpeptideonto thenewaminoacidthat has just been brought in
What does elongation factor (EF1) and the hydrolysis of GTP do?
Allows the aminoacyl tRNA to bind to the aminoacyl site on the ribosome
What enzyme, energy source and elongation factor does translocation factor require?
Enzyme translocase
EF 2
GTP hydrolysis
What are polysomes?
In most cells, each mRNA has quite a few ribosomes attached so one message is synthesising multiple parts of the protein as the same time called polysomes
What are the three STOP codons?
UAA, UGA, UAG
Explain termination
The ribosome reaches the STOP codon.
When this happens, release factor protein recognises this and using GTP energy, hydrolyses the bond holding the tRNA to the peptide chain and it drops off.
How many reading frames are there in mRNA?
3
The codons in mRNA are read in a continuous batch so there are three separate reading frame
Each reading frame translates to a completely different peptide sequence
Only ONE frame is used for each mRNA, this is determined by the location of the AUG start codon.
What changes reading frames?
Deletion or insertions of bases due to mutations in DNA
Compare bacterial and mammalian ribosomes
Prokaryotic are 70s (50s and 30s)
Eukaryotic are 80s (60s and 40s)
What is the significance of ribosomes in eukaryotic and prokaryotic cells being different?
It means that certain substances can be used to target only specific ribosomes and hence affect only protein synthesis in prokaryotes without effecting eukaryotic protein synthesis.
e.g. tetracycline
What does streptomycin do?
It acts on the small ribosomal subunit.
Inhibits initiation - misreading of genetic code
What does tetracyclines do?
It acts on the small ribosomal subunit
Inhibition of aminoacyl tRNA binding to ribosome
What does chloramphenicol do?
It acts on the large ribosomal subunit
Inhibition of peptidyl transferase activity
What does erthromycin do?
It acts on large ribosomal subunit
Inhibition of translocation
What are the limitations of antibiotics on protein synthesis?
- Antibodies have no action on viruses (viruses use host’s protein synthesis machinery)
- Encourages bacteria to develop resistance against bacteria
(e. g. changing the ribosome, destroying antibiotic through enzyme) - Long term antibiotics can impair energy production plus drug metabolism (mitochondria are very similar to prokaryotic ribosomes so antibiotics that affect bacteria protein synthesis have an impact on mitochondrial protein synthesis)
