Theme 2- Module 3 (Translation) Flashcards
Is each tRNA molecule identical? How does this aid in their function?
No, bc of this, they can translate a specific RNA codon into a specific amino acid
Describe the structure of a tRNA molecule?
Single RNA strand, 70-90 nucleotides in length
Large degree of complementarity. Many stretches of hydrogen bonding between complementary nucleotide bases –> Four double helix segments and three loops
Folds into a roughly L-shaped 3D structure
What is the “anticodon region” of tRNA?
Specific nucleotide triplet that is complementary to a specific mRNA codon that codes for a specific amino acid
The anticodons in tRNA are written in which direction relative to the mRNA codons?
3’–>5’ direction but align to the mRNA codons in the 5’–>3’ dir
What is located at the 3’ end of a tRNA molecule?
Protruding amino acid attachment site that is made up of a single CCA nucleotide sequence
Terminal A (adenine) = actual point of attachment for an amino acid during tRNA molec activation
What is aminoacyl tRNA synthase?
Enzyme that matches/activates the tRNA with specific amino acids
Are all aminoacyl tRNA synthases the same?
No, each of them is specific to the amino acid and tRNA that it will bind to
Describe the process of tRNA activation
The active site of synthase enzymes recognise the antocodon end of the tRNA and the region of the amino acid attachment site
Once bound to active site, these enzymes catalyze the covalent attachment
This leads to a charged tRNA or an aminoacyl being released from the enzyme (and can now deliver amino acids)
Does activating tRNA require energy?
Yes
How many aminoacyl tRNA synthases are there?
20 – one for each amino acid
True or false: mRNA can code for 64 possible codons. This means that there are 64 tRNA molecules to match each codon
False
There are 45 tRNA molecules which means that some are able to bind to more than one codon
What does “wobble” mean in terms of tRNA?
Explains why some tRNA molecules able to bind to more than one codon
While the first base (5’ end) of the codon will bind to the last base (3’ end of anticodon), there is a greater flexibility for base pairing between the third nucleotide of the codon and the corresponding base of a tRNA anticodon
How do prokaryotes initiate translation?
Initiation complex binds at Shine-Dalgarno sequences (ribosome binding sites)
(prok don’t have a 5’ cap)
How do eukaryotes initiate translation?
Translation initiation complex forms towards 5’ cap of mRNA and scans it until an AUG codon is encountered
Where are Shine-Dalgarno sequences usually located on the mRNA?
A few bases upstream of AUG
Why is the mRNA in prokaryotes polycistronic (able to code for more than one peptide with the same mRNA strand)?
Shine-Dalgarno sequences can occur multiple times and translation can happen at all of them simultaneously.
Prok have functionally related genes grouped together along its DNA; transcribed together
The initiation of translation req the assembly of various components, such as….
- Two ribosomal subunits
- mRNA (to be translated)
- charged tRNA methionine (anticodon for AUG)
- initiation factors
Describe the assembly of the translation initiation complex in eukaryotes
Initiation factors bind to 5’ cap
Recruits small ribosomal subunit
Other initiation factors bind to charged tRNA that has methionine
This complex moves along mRNA until an AUG is encountered. When it finds the AUG, the large ribosomal subunit binds and so does the next charged tRNA molecule
Where does the large ribosomal subunit get the energy to bind to the rest of the initiation complex?
GTP hydrolysis
When the partially assembled initiation complex is moving along, trying to find the start codon, which direction does it move in?
5’ to 3’
Once the the initiation complex is assembled, what happens to the initiation factors?
They are released
True or false: all amino acids are synthesized from the amino end to the carboxyl end
True
Which amino acid is the first one to be found at the amino end of the polypeptide?
Methionine
Which site of the ribosome do all incoming charged tRNA molecules enter and bind to prior to each amino acid being incorporated into growing polypeptide chain?
Aminoacyl
What are the three sites of the ribosomal subunit (in order from entrance to exit)?
Aminoacyl
Peptidyl
Exit
Which site will methionine be located in at the start of translation (unlike all of the other amino acids)?
Peptidyl
How are incoming charged tRNA molecules able to enter and bind to the sites on the ribosome (considering the amino acid hasn’t even been added yet)?
Seq of mRNA coding is read by the ribosome in successive, nonoverlapping groups of three nucleotides
Each charged tRNA is delivered to the ribosome with a…?
GTP-bound elongation factor
What happens to the GTP-bound elongation factor as the tRNA enters the A-site on the ribosome?
When correct codon-anticodon pairing is made, GTP turns to GDP and the aminoacyl end of tRNA is released from the elongation factor
What is the max number of charged tRNA molecules that can be present within the ribosome at any given time?
2
The E site does not have any charged tRNA
Following the binding of a charged aminoacyl tRNA, what happens?
There is a conformational change in the ribosomal RNA that allows for a peptidyl-transferase reaction to occur between the amino acids to form a polypeptide chain
Describe the peptidyl transferase reaction that occurs between the amino acid from the P-site and the amino acid from the A-site
Forms a condensation rxn as a peptide bond, and a transfer of the growing polypeptide chain onto the tRNA that is in the A-site
What allows the ribosome to continue to translocate along the length of the mRNA molecule?
GTP-bound elongation factors cause the deacylated tRNA to move from the P-site to the E-site. When the next tRNA enters the A-site, the deacylated tRNA is released from the E-site
What happens once the ribosome reaches the STOP codon?
GTP-bound release factors bind to the A-site and catalyse the hydrolysis of the bond between the terminal amino-acid in the polypeptide and the tRNA in the P-site
Further GTP hydrolysis enables dissociation of translation complex
Describe the “one-gene-one-enzyme” hypothesis that was established by George Beadle and Edward Tatum
Based on the fact that Neurospora crassa can grow on a minimal medium (that contains only simple sugars, etc.).
Hypo: must have some enzymes produced by a specific gene that convert these simple substances into the amino acids and vitamins that are needed for growth
Short form: each gene contains the info needed to make each enzyme
How do they know that neurospora are able to synthesize their own arginine (amino acid)?
Bc they are able to grow well in a medium that lacks arginine, therefore, they must be able to make it themselves
What is the metabolic pathway for argenine synthesis?
Precursor
Ornithine
Citrulline
Arginine
In argenine synthesis, the transition between steps requires what?
A separate enzyme for each step
Describe the procedure of Srb and Horowitz’ experiment that tested the one-gene-one-enzyme hypo
Performed a genetic screen of radiation treated neurospora to det whether there were specific genes that produced each of the three enzymes that are needed in argenine synthesis
Raised these colonies on a medium that was supplemented with either nothing (precursor), ornithine, citrulline, or arginine (to prevent mutation)
What were the results of Srb and Horowitz’ experiment?
Medium with argenine —> growth
Other mediums –> no growth
In the mediums with ornithine and citrulline, there was an inhibition of growth
Why was the surface of the liquid in the test tube in Srb and Horowitz’ experiment slanted?
To allow room for growth
What conclusions were made from Srb and Horowitz’ experiment?
Radiation produced mutations in the genes that encode for the necessary enzymes for the production of argenine
Found three mutants: arg1, arg2, arg3. Arg1 cells lack enzyme needed to make precursor into ornithine. Arg 2 lack enzymes needed to make ornithine into citrulline and so on…
Why is the one-gene-one-enzyme hypo usually called the “one gene, one polypeptide” hypo now?
Bc they found out later that genes dictate structure of all proteins
What is the human proteome?
Rep the full number of proteins that are expressed by all of the hereditary info in our DNA (aka our genome)
There are over 1 million proteins in our bodies, but only 20-25000 genes that encode for them. What does this mean?
More than one protein can be produced from a single gene
A mutation in arg2 prevents argenine synthesis. The defect can be rescued by the addition of which molecule?
Citrulline
