Protein Synthesis Flashcards
What are the key features of the Genetic Code?
1) The genetic code is universal: All organisms use the same genetic code
2) The genetic code is degenerate: Multiple codons encode for the same amino acid. e.g. GGU, GGC, GGA and GGG all encode for glycine. Degeneracy is usually at the third nucleotide or the wobble base.
3) Some codons have multiple functions: AUG for example encodes for Methionine but it is also the START codon. UAA, UAG, and UGA all are STOP codons.
There are 64 total triplets of nucleotides but only 20 amino acids that are coded for so we clearly have more than one triplet coding for a specific amino acid.
Codons for the same amino acid tend to share the same first two nucleotides (but this is not always true). Also, not every amino acid has more than one codon that codes for it.
Identify features of an mRNA that can affect its translation.
An mRNA needs to contain a 5’-cap and a Polyadenylated tail in order to be read as a “good” mRNA. Also, the mRNA must have the correct reading frame. The sequence of nucleotides are read 5’ to 3’ in translation in consecutive sets of 3 nucleotides. Thus, there are hypothetically 3 reading frames that exist and thus the same mRNA could give 3 different polypeptides. In reality, only one of these reading frames contains the actual message and if the wrong reading frame is read, there tends to be a lot of sporadic STOP codons.
Translation START codons
AUG
Codes for Methionine
Translation STOP codons
UAA, UAG and UGA
They do NOT code for amino acids.
Degenerate
Multiple codons code for the same amino acid
4 Major Steps in Translation
1) Charging of the tRNA: Placing of the amino acid
2) Initiation
3) Elongation
4) Termination
Transfer RNA (tRNA)
It is synthesized by RNA Pol III. Also, it is synthesized as a precursor, it will be trimmed, spliced, and modified. There are over 50 different modifications for tRNA and one out of every 10 bases will be modified. This modification is thought to be important for the final folding of the tRNA and to get the anticodon region in the right position to get good base pairing with the codon.
tRNA contains some unusual bases that are created during the modification of it such as pseudouridine and dihydrouridine which are derive amino acids from uracil.
There are 2 areas of interest:
1) anticodon
2) 3’ acceptor stem
Anticodon
it is a region on the tRNA that is 3 nucleotides long and is responsible for base pairing with the codon to ensure correct amino acid addition to the newly synthesized polypeptide chain. The pairing proceeds from the 5’ end of the codon. Once the first two positions are paired, the exact base pairing of the third (wobble position) is less critical. Wobble positions allow some tRNAs to recognize more than one mRNA codon making it possible for less than 64 tRNAs to recognize all 64 codons. The wobble position in terms of the tRNA though is in the FIRST position, but we call it the third position in regards to the mRNA.
READ mRNA 5’ to 3’ NOW!!!
3’ Acceptor Stem
This is where the amino acid is physically attached to. Aminoacyl-tRNA synthetase catalyzes the two step activation reaction of the tRNA.
Aminoacyl-tRNA Synthetase
Catalyzes the two step activation reaction of the tRNA. It catalyzes the addition of the amino acid to the 3’ acceptor site. Eukaryotes have a DIFFERENT aminoacyl-synthetase for EACH amino acid whereas in prokaryotes, each synthetase couples more than one amino acid. The aminoacyl-synthetase is also crucial in regards to proofreading. It is the last check to make sure that the correct amino acid was added to the tRNA.
Explain the differences between prokaryotic and eukaryotic cells with respect to aminoacyl-synthetases.
Eukaryotes have DIFFERENT aminoacyl-synthetases for EACH amino acid.
Prokaryotes have ONE synthetase which couples for more than one amino acid.
Identify important structural features in tRNA molecules, including features that contribute to fidelity in protein synthesis.
Two major structural features are:
1) anticodon
2) 3’ acceptor site
the anticodon ensures correct base pairing with the codon sequence of the mRNA, specifically with the first two nucleotides. It is what reads the codon to see what amino acid is supposed to be added. If the sequences don’t match, they will not bind which gives it fidelity.
The 3’ Acceptor site adds to the fidelity because it binds specifically to the correct amino acid via the assistance of the Aminoacyl-tRNA Synthetase. The specific aminoacyl-tRNA Synthetase has an editing site that ensures the correct amino acid is bound.
Draw an aminoacylated tRNA molecule and diagram the reactions that produced it.
View reaction in handout. IT IS A 2 STEP ACTIVATION OF THE tRNA. Basically, carboxy-end’s OH reacts with ATP, adding AMP to the O, losing 2 inorganic phosphates and the H. Then, the now adenylated amino acid (AMP-Amino acid) reacts with the OH of the 3’ acceptor site, giving off AMP and resulting in the amino acid bound to the tRNA via a high energy ester-linkage, thus activating it. This energy is important because it plays a crucial role in providing the energy to create the polypeptide chain. Overall it is using the energy from ATP hydrolysis to add the amino acid to the tRNA.
Explain the role of editing by some aminoacyl-tRNA Synthetases in determining the fidelity of protein synthesis.
The synthetase is actually sensing that the correct amino acid is bound to the correct tRNA. It plays a crucial editing step to ensure high fidelity because it is one of the only steps to do this. The synthetase is able to do this so well because it is physically interacting with both the amino acid and the tRNA. It is interacting with the various parts of the tRNA and will make sure the 3 anticodons are correct and match this amino acid. However, some anticodons that code for the same amino acid are very different so it also relies on other parts of the tRNA as well.
What are the two “adaptors” that the genetic code is translated by means of?
1) The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino acid to its corresponding tRNA
2) The second adaptor is the tRNA molecule itself, whose anticodon forms base pairs with the appropriate codon on the mRNA.
These two occur directly after one another and an error i neither step would cause the wrong amino acid to be incorporated into the protein chain.
“Second Genetic Code”
The recognition of the correct tRNA by the aminoacyl-tRNA Synthetase
Aminoacyl-tRNA Synthetase and tRNA interactions to ensure correct tRNA and amino acid binding
The aminoacyl-tRNA Synthetase recognizes multiple regions on the tRNA to ensure that it is indeed the correct amino acid that is binding. The 2 most important and most commonly used regions are the anticodon loop and the 3’ acceptor stem. These are particularly important in controlling the specificity of the aminoacylation reaction. However, some aminoacyl-tRNA synthetases use other regions on the tRNA to distinguish some tRNAs that are difficult to tell apart. There are many regions on the tRNA that the synthetase recognizes that are crucial for this high fidelity.
Explain the Proofreading activity of the aminoacyl-tRNA Synthetase
The aminoacyl-tRNA synthetases that need to distinguish between similar amino acids have an added proofreading mechanism to ensure they attach the correct amino acid to the correct tRNA. The correct amino acid has the highest affinity for the synthesis site, however, sometimes errors occur. Thus, the aminoacyl-tRNA synthetase has an editing site in which if the WRONG amino acid has been bound to the tRNA, it will enter the editing site. Then, it will be cleaved off and the correct amino acid will then be added. If the amino acid is correct, it cannot enter the editing site. The accuracy is 1 in 40,000 and this is where most of the proofreading is because once the amino acid is added, there is no going back.
Shine-Delgarno Sequence
This is found in PROKARYOTES. It is in front or upstream of the AUG start codon. NOT ALL AUGs HAVE THE SEQUENCE IN FRONT OF THEM. it is a 3-9 PURINE RICH base pair sequence that is just upstream (~10 nucleotides) of the START codon (AUG) and the small ribosomal subunit base pairs directly with it and then the ribosome assembly takes place at the AUG start codon. The shine-delgarno sequence is complementary to the 3’ end of the 16S-ribosomal RNA in the prokaryotic 30S ribosomal subunit. The AUG begins in the P-site of the ribosome and this is where the first tRNA will begin at to give the correct reading frame. fMet-tRNA or formyl-Methionine is what binds first to the AUG and it is a special initiation tRNA with a modified methionine. It is only found in prokaryotes.
List the major steps in protein synthesis
1) Charging of the tRNA
2) Initiation
3) Elongation
4) Termination
Describe the interaction between the mRNA codon and the tRNA anticodon paying particular attention to the 5’ and 3’ ends of both strands
The mRNA is being read 5’ to 3’, thus, the tRNA’s anticodon will bind to the mRNA antiparallel (3’ with 5’ and 5’ with 3’). first site of the tRNA is the 3’ end of the tRNA, which binds to the first nucleotide (5’ end) of the codon. Then the second site binds to the middle of the codon and the last site, the 5’ end of the tRNA and the 3’ end of the mRNA is the wobble position.
Describe the Wobble Phenomenon and explain its relationship to the existence of degeneracy in the genetic code.
The wobble phenomenon basically states that the third nucleotide or the third position of the codon (first of the tRNA when reading it 5’ to 3’) can vary, or “wobble”. In other words, the nucleotide itself can vary from what it should be to still give the correct amino acid since most of the base pairing is mainly dependent on the first two codon positions. In other words, in positions 1 and 2, conventional base pairing MUST take place (A with U and C with G) but in the third position we don’t need this. This relates to degeneracy because when one looks at most codon sequences for amino acids, the first two positions of the codon are mainly the same and the third position is the difference.
Describe the functions of the large and small ribosomal subunits in protein synthesis.
The small ribosomal subunit is responsible for establishing the reading frame and helps to initiate protein synthesis. Then, the large ribosomal subunit will come it and it is what has the catalytic activity to start forming the polypeptide chain and forms the peptide bond.
What are the initiation steps in prokaryotes?
1) IF-3, the 30S ribosomal subunit (small subunit), and the mRNA come together with the small ribosomal subunit binding to the shine-delgarno purine rich 3-9 nucleotide sequence, placing the ribosome ON the AUG start site (will be in the P-site of large subunit)
2) IF-2 with a bound GTP and the initiator tRNA with a formyl-methionine will come together at the AUG start site. The energy from the bound GTP is critical for initiation.
3) tRNA then binds to the AUG with the IF-2-GTP. The GTP is then hydrolyzed and this recruits the 50S large ribosomal subunit.
4) Elongation occurs
KNOW IF-2 HAS GTP BOUND TO IT