Bacterial Translation Flashcards
What does transcription create from the coding region of a gene or operon?
An mRNA copy of the coding region
What direction does the synthesis of DNA and RNA occur in?
The 5’ to 3’ direction
What sequence strand does RNA polymerase read?
The template strand
When RNA polymerase reads the template strand, what rules are used and what does it create?
Base pairing rules
A copy of the sense (coding) strand from the 5’ to 3’ end
What are the base pairing rules for bacterial translation?
G pairs with C
A pairs with U
What is the first sequence of the mRNA and what does it assist in?
The leader (5’-UTR) and it assists in the initiation of the translating process
After the leader, the sequence of mRNA is read in codons, which are?
Groups of 3 nucleotides
What is the start codon?
Almost always AUG and is the first codon. It sets the reading frame of the mRNA.
Define the reading frame
The division of a sequence of an RNA molecule into consecutive, non-overlapping sets of 3 nucleotides.
What does each codon specify?
One of the 20 standard amino acids
When the ribosome glides along the reading frame what happens?
It uses the sequences of codons to synthesize a cha8n of amino acids (a polypeptide)
When does the ribosome stop reading the frame?
When it reaches a stop codon.
What are the 3 stop codons?
UAG (amber)
UAA (ochre)
UGA (opal)
What is the translation of the RNA codon to amino acids performed by?
Transfer RNA (tRNA) molecules
What is each molecule of tRNA and what is it able to do?
Each is a single stranded RNA able to form base pairs with itself to fold into a 3D structure.
What is the job of tRNA?
Form base pairs with the codons of the mRNA and carry the amino acids that matches that codon into the ribosomes.
What are the 4 functional areas of the tRNA? Explain each.
- The anticodon loop: forms base pairs with the correct codon on the mRNA in the ribosome.
- Acceptor stem: where the correct amino acid is attached.
- T-psi-C arm: allows the tRNA to bind to the ribosome itself
- D arm: allows the tRNA to bind to the enzyme that attaches its amino acid
What enzyme is each tRNA associated with?
Aminoacyl-tRNA synthetase which attaches to the correct tRNA by binding to the anticodon loop and D arm.
What does aminoacyl-tRNA Synthetase do once bound?
Uses the energy within a molecule of ATP to attach the correct amino acid to the acceptor stem using a covalent bond.
Where does translation take place?
In the 70S ribosome.
What is the 70S ribosome made of?
A large ribosomal subunit (50S) built from 34 different proteins and 2 mRNA molecules (the 5S and the 23S ribosomal RNAs (rRNA))
The small ribosomal subunit (30S) built from 21 different proteins and 1 RNA molecule (16S ribosomal unit)
What are the 3 internal binding sites of the ribosome for tRNA?
- The acceptor (A site)
- Peptidyl (P site) is where the peptide bond is formed between amino acids
- Exit (E site) where tRNA leaves the ribosome
What makes the tRNA an aminoacyl-tRNA?
When the correct amino acid is attached to the acceptor stem.
What are the 3 phases of bacterial translation? Explain them.
- Initiation: ribosome binds to and assembles round the mRNA to be translated.
- Elongation: the ribosome reads the mRNA sequence and creates the polypeptide chain.
- Termination: the ribosome reads the stop codon and then disassembles itself.
Initiation of translation requires the assembly of the initiation complex. What 4 molecules does the complex consist of?
- The small ribosomal subunit
- The mRNA to be translated
- A tRNA molecule with a special amino acid called N-formula-methionine (fMet)
- Three initiation factor proteins called IF-1, IF-2 and IF-3
In the first step of translation initiation, what subunit does the mRNA needing to be translated bind to? What does the mRNA leader sequence contain and what does it allow?
30S subunit
Contains a series of nucleotides called the shine dalgarno sequence that forms base pairs with the 16S ribosomal RNA.
What does the interaction between the 16S and the leader sequence do?
Precisely positions the start codon in the P site of the ribosome.
What is happening elsewhere when the 16S and leader are positioning the start codon?
The initiation factor IF-3 is bound to the 30S subunit preventing the 30S subunit to prematurely bind to the 50S subunit.
What does IF-2 bind to and what does that allow?
It binds to GTP and the tRNA containing fMet. All together these will base pair with the AUG (start codon) on the mRNA
Once the fMet-tRNA is base paired with the mRNA bound to the 30S subunit, what happens? What complex is formed?
IF-1 binds to the 30S subunit causing IF-3 to unbind. This is called the 30S initiation complex.
What happens once the 30S initiation complex is formed?
The 50S subunit binds causing IF-2 to hydrolysis its GTP.
What doesthe energy from GTP hydrolysis cause?
IF-1 and IF-2 to unbind from the 30S and the 2 ribosomal subunits become “locked” together and initiation is complete.
Initiation is complete and elongation is beginning. What are the 3 events that repeat in a cycle?
- Aminoacyl-tRNA binding.
- Transpeptidation
- Translocation
Explain the aminoacyl-tRNA binding during elongation.
Elongation factor Tu (EF-Tu) (a protein) binds to the tRNA carrying the correct amino acid. Then EF-Tu inserts the tRNA molecule into the A site of the ribosome using the energy provided by GTP Hydrolysis.
Explain transpeptidation of elongation.
Peptide bond is formed between the amino acid in the A site and the polypeptide in the P site. This reaction is performed by the 23S ribosomal RNA of the 50S (large) subunit.
Explain translocation of elongation.
Entire ribosome shifts to the next codon, shifting the tRNAs into the next site. This opens the A site for the next amino acid. Any tRNAs in the E site are ejected from ribosome.
What is translocation performed by ?
EF-G. Moving the ribosome costs energy which EF-G provides using GTP hydrolysis.
What happens when translocation is complete?
Cycle repeats until a stop codon is reached.
Explain termination of bacterial translation.
Requires a release factor (a protein) which does 2 things:
1. Hydrolyzes the covalent bond connecting the polypeptide to the tRNA in the P site, releasing the completed protein from the ribosome.
2. Causes the complete disassembly of the ribosome.
To translate a protein again, what must happen?
Start over with the translation initiation.
