Unit 8 Flashcards
Given that there are four different bases, what is the minimum number of nucleotides per amino acid required to specify all 20 amino acids?
3 nucleotides because 4^3 is the first number that is above
Define codon
A sequence of 3 adjacent nucleotides in a nucleic acid that codes for a specific amino acid
Is the code overlapping or non-overlapping?
The code is non-overlapping
Distinguish between a nonoverlapping code and overlapping code
Non Overlapping:
AUA - CGA - GUC
Overlaping:
AUA CGA GUC
UAC
ACC
Define reading frame
A specific way of dividing a sequence of nucleotides into a series of consecutive, non-overlapping codons during the process of translation
Are individual codewords delimited, as for example words in a sentence are delimited by spaces?
No. There is no punctuation between codons for successive amino acids.
What establishes the reading frame?
It is established by the start of translation of an mRNA molecule. In mRNA, this starting point is typically the first AUG (start codon) which encodes methionine
Given a sequence of RNA, an indication of the correct reading frame, and the genetic code, deduce the relevant polypeptide sequence encoded in the reading frame
- The third base of each codon plays a letter role in specifying an amino acid than the first two
- The three termination codons are shaded in red and the initiation codon, AUG, in green
What are the expected consequences of a one or two base addition or deletion within the above sequence?
- Inserting or deleting one base pair alters the sequence of all amino acids coded by the mRNA following the change
- However, if there is a combined insertion and deletion mutation, it will only affect some amino acids but eventually the reading frame will be restored to the correct amino acid
Why are such additions or deletions (when they occur in DNA) called frameshift mutations?
Because they alter the reading frame
Initiation codon
AUG is the most common signal for the beginning of a polypeptide in all cells, in addition to coding for Met residues in internal positions of polypeptides
Termination codon
UAA, UGA, UAG; in protein synthesis, these codons signal termination of polypeptide synthesis and do not code for any known amino acid
Open reading frame
A reading frame without a termination codon among 50 or more consecutive codons are referred to as an open reading frame. But it does have a stop codon at the end
What does degeneracy refer to in the context of coding?
Degeneracy means that an amino acid may be specified by more than one codon
ex. Arg is coded by 6 codons
Describe how aminoacyl tRNA interact with mRNA
- Transfer RNAs base-pair with mRNA codons at a three-base sequence on the tRNA called the anticodon. The first base of the codon in mRNA (read in the 5’ to 3’ direction) pairs with the third base of the anticodon
- Alignment of the two RNAs are antiparallel
*draw this out to see if you can do it
Is the genetic code almost universal
Genetic code is not quite as universal as once believed, but that is flexibility is severely constrained. The variations are obviously derivatives of the cellular code, and no example of a completely different code has been found
*basically the differences are that some codons do not code for the same amino acids in other animals
The genetic code has been conserved through evolutionary time because it is resistant to the deleterious effects of mutations. Discuss and give an example of, how the genetic code protects against missense mutations at both the 5’ and 3’ nucleotide positions in the codon
- A mutation in the first position of the codon will usually produce an amino acid coding change, but the change often results in an amino acid with similar chemical properties
(ex. Val’s codon is GUU. A change to AUU would substitute Ile for Val, but they are both still hydrophobic) - A mutation in the last position is also protected due to the wobble hypothesis. The wobble hypothesis states that only the first two amino acids bind tightly, so the third can be variable
Discuss the meaning of “wobble”
- The wobble (or third) base of the codon contributes to specificity, but because it pairs only loosely with its corresponding base in the anticodon, it permits rapid dissociation of the tRNA from its codon during protein synthesis
- If all three bases of a codon engaged in strong pairing with the 3 bases of the anticodon, tRNAs would dissociate too slowly and this would limit the rate of protein synthesis
What is degeneracy a feature of?
The genetic code (some amino acids are encoded for by more than one codon)
What is wobble a feature of?
The tRNA (some tRNAs can bind to more than one codon)
Show how guide RNAs can be used as templates to direct the addition or removal of specific nucleotides from an mRNA transcript
- RNA editing is a post-transcriptional editing process that can alter the meaning of one or more codons during translation
- The insertions require a special class of guide RNAs that act as a template for the editing process
- The base pairing between the initial transcript and the guide RNA includes several G-U base pairs which are common in RNA molecules
*the guide RNA will not be able to form strong base pairs with mismatched amino acids so amino acids that can base pair strongly with the guide RNA will be inserted in
Show how deaminases can alter specific nucleotides in an mRNA transcript.
- The adenosine deamination reactions are carried out by adenosine deaminases that act as RNA (ADARs). Turns Adenosine into Inosine
- The Cytidine deaminations (to Uridine) are carried out by apoB mRNA editing catalytic peptide (APOBEC)
*deamination means the top NH2 is turning into a C=O bond
How does RNA editing by deaminases can regulate the production of two related but different products from a single transcript in a tissue specific manner
RNA editing deaminates a specific cytidine to uridine (only occurring in the intestine) changing a Gln code to a stop codon and producting a truncated protein (shortened protein)
Summarize the key events that occur during each of the following stages of protein synthesis
- Activation of amino acids
- Initiation of protein synthesis
- Elongation
- Termination and ribosome recycling
- Folding and post-translational processing
Activation of amino acids
The tRNA is aminoacylated
Initiation of protein synthesis
the mRNA and the aminoacylated tRNA bind to the small ribosomal subunit. The large subunit then binds
Elongation
Successive cycles of aminoacyl-tRNA binding and peptide bond formation occur until the ribosome reaches a stop codon
Termination and ribosome recycling
Translation stops when a stop codon is encountered. The mRNA and protein dissociate, and the ribosomal subunits are recycled
Folding and post-translational processing
Protein folding and post-translational processing
Discuss the molecular composition and 3D conformation of the ribosome
- Ribosomal subunits can be broken down into their RNA and protein components
- Composed of 2 subunits (large and small) 30S and 50S
- The cleft between the 50S and 30S is where the protein synthesis occurs
Illustrate that RNA (in addition to proteins) contributes to this 3D structure of ribosomes
- The folding pattern of the rRNAs are highly conserved in all organisms, particularly the regions implicated in key function
- There is a conservation of secondary structure in the small subunit rRNAs from 3 domains of life
Discuss the ribosome in the context of the RNA world hypothesis
- Ribosome is ribozyme so it has a catalytic role but the RNA in the ribosome is also responsible for storing information. These are important features of RNA as seen in the RNA World hyothesis
Why are tRNAs referred to as “adaptors”
tRNAs are referred to as adaptors because they translate the language of nucleic acids into the language of proteins
Are tRNA synthetastes specific for amino acids?
Every tRNA synthetase is specific for only ONE amino acid (they check this by seeing if it fits in the protein). Since multiple codons can encode for an amino acid, the tRNAs (which is the same as codons) are specific for an amino acid but the amino acid isn’t specific for one individual codon/tRNA
Make sure you can draw and label the structure of tRNA
The anticodon arm (or loop)
Contains the anticodon
The TYc arm
- Contains ribothymine (T) and pseudouridine (trident). The D and TYC arms contribute important interactions for the overall folding of tRNA molecules, and the
- TYC arm interacts with the large subunit rRNA
The dihydrouracil arm (D arm)
- Contains the nucleotide dihydrouridine
- Contributes important interactions for overall folding of tRNA molecules
The 3’ (CCA) and 5’ ends of the RNA molecule
Most tRNAs have a guanylate (pG) residue at the 5’ end and all have the trinucleotide sequence CCA at teh 3’ end
The position at which the amino acid is attached
The aminoacyl group is esterified to the 3’ position of the terminal A residue
Regions that are hydrogen bonded
When drawn in 2D, all tRNAs have hydrogen-bonding patterns that form a cloverleaf structure with 3 arms
What are the two steps for how the activation of an amino acid for protein synthesis takes place. Show the structures of the reactants, intermediates, and the products
*draw this out
- Formation of the aminocacyl-adenylate
- Aminoacyl group is transferred to tRNA
Name the class of enzymes that loads amino acids onto tRNAs
aminoacyl-tRNA synthetases
Name the linkage that is broken in ATP
Phosphoanhydride linkage
Name the linkage that is formed in the activated intermediate which is then broken in the final stage of the reaction
Mixed anhydride linkage
Name the linkage that is ultimately formed between the amino acid and the tRNA
Ester linkage
What is the other product of this series of reactions? What happens to this product?
AMP
It’s phosphorylated to ATP
Write a balanced word equation for the sum of the activation and transfer steps, including hydrolysis of PPi
Amino acid + tRNA + ATP (tRNA synthetase –>) Aminoacyl-tRNA + AMP + PPi
There is a cost of two high energy anhydride bonds in the synthesis of an aminoacyl-tRNA? Account for this cost
*essentially this question is asking which bonds had to break which are a cost of the reaction
Phosphate breaking down
Mixed anhydride breaking
Name two other synthetic processes you have studied in which pyrophosphate is generated. What is the significance of the fact that all cells possess pyrophosphatases that catalyze the hydrolysis reaction PPi +H20 –> 2Pi
- DNA Replication and RNA transcription
- The significance is that the hydrolysis of the pyrophosphate drives the reaction forward for these processes due to Le Chatelier’s Principle
Discuss how Ile-tRNA synthetase distinguishes between the Ile and Val at high fidelity
The Ile-tRNA has a proofreading function to make sure the right amino acid is added to the tRNA
1. In the initial binding of the amino acid to the enzyme, if it’s not the correct amino acid, it won’t bind to the enzyme
2. If by chance, the amino acids are very similar like Val and Ile, it can bypass teh first filter and get AMP added to it (the first step in the activation of amino acids). It will then be moved to the secondary site. If the molecule is small enough, it will fit into the site and be hydrolyzed to separate the Val and AmP. Isoleucine should not fit in this site, so if something does, it knows that it is not Ile and wil hydrolyze whatever it is so it’s inactive
DRAW OUT WHAT IS HAPPENING AT THE DIFFERENT PARTS OF THE AMINOACYL-TRNA SYNTHETASE SO YOU KNOW WHERE STEP 1 and STEP 2 ARE OCCURING
What is the second genetic code
An individual aminoacyl-tRNA synthetase must be specific not only for a single amino acid but for certain tRNAs as well. Discriminating among dozens of tRNAs is just as important for the overall fidelity of protein biosynthesis as is distinguishing among amino acids
Discuss the key events in translation initiation
- The 30S subunit binds IF1 and IF3, then the mRNA. The Shine-Dalagarno sequence on the mRNA interacts with the 16S rRNA
- IF2-GTP binds the 30S subunit adn recruits fMet-tRNA which base pairs with the start codon
- The 50S subunit associates, IF2 hydrolyzes FTP, adn IF1, IF2, and IF3 dissociate, leaving the 70S initiation complex
What is the Shine-Dalgarno sequence? Explain its significance in prokaryotic translation initiation
- A sequence in an mRNA that is important for binding bacterial ribosomes
- The Shine-Dalgarno sequence interaction with the 3’ end of the 16S rRNA positions the initiating (5’) AUG sequence of the mRNA in the precise position on the 30S subunit where it is required for initiation of translation (P site)
Account for the cost of one high energy bond used when IF-2 delivers an aminoacyl tRNA to the P site during translation initiation
The hydrolysis of GTP to GDP +Pi
Compare the initiation of translation in prokaryotes and eukaryotes
- Translation is generally similar in eukaryotic and bacterial cells, most of the significant differences are in the number of components and mechanistic details
- Eukaryotes:
- Different, larger ribosomes
- 12 initiation
- mRNA is bound to 5’ cap and 3’ poly A tail, circulating the mRNA
- No shine-dalgarno sequence
*remember eukaryotic cells are monocistronic so they don’t need shine-dalgarno while prokaryotic cells are polycistronic
Discuss the role of EF-Tus in delivering AA-tRNAs to the A site during elongation. Account for the one high energy bond used by EF-Tu.
- The incoming aminoacyl-tRNA binds to the GTP bound EF-Tu. The resulting aminoacyl-tRNA - EF-Tu-GTP complex binds to the A site of the 70S initiation complex
- The GTP is hydrolyzed and the EF-Tu-GDP complex is released
- The aminoacyl tRNA in the A site changes conformation as a result of the hydrolysis energy to put itself in a position where the amino acid could form a peptide bond
What is the role of EF-Ts
Phosphorylate the EF-Tu-GDP into GTP
Name the enzyme and point out the nucleophilic attack mechanism catalyzed by this enzyme. Is this reaction catalyzed by a protein enzyme or a ribozyme
- Peptidyl transferase
- Ribozyme
Draw out the nucleophilic attack for the peptide bond formation
Does protein synthesis run from N to C terminal end of C to N
N to C
Note the position of the peptidyl tRNA after peptide bond formation
On the tRNA in the A site
Note: No additional input of energy is required for peptide bond formation because the energy required is released when the peptidyl-tRNA ester bond is broken
Discuss translocation. Account for the one high energy anhydride bond that is used to translocated the peptidyl tRNA from the A ssite to the P site
- The ribosome moves one codon towards the 3’ end of the mRNA, using energy provided by hydrolysis of GTP bound EF-G (translocase)
- The dipeptidyl-tRNA is now entirely in the P site, leaving the A site open for an incoming aminoacyl tRNA. The unchanged tRNA later dissociates from the E site, and the elongation cycle begins again
Discuss the role of EF-Tu in proofreading
- Both the EF-Tu-GTP and EF-Tu-GDP complexes exist for a few milliseconds before they dissociate. These two intervals provide opportunities for the codon-anticodon interactions to be proofread
- Incorrect aminoacyl-tRNAs normally dissociate from the A site during one of these periods (because the codes won’t match so it will dissociate)
Account for the one high energy bond used in termination and discuss termination
- For termination, the stop codon is read in the P site which recruits a release factor. Release factor hydrolyzes the polypeptide from the tRNA. The EF-G -GTP uses hydrolysis to move the ribosome over one but this ends up dissociating the complex
Folding and Processing: Describe some ways that newly synthesized polypeptides are processed following their syntehsis
- During or after synthesis, the polypeptide progressively assumes its native conformation, with the formation of appropriate hydrogen bonds and van Der Waals and inic interactions through the hydrophobic effect
- Protein chaperones help in folding, some other proteins require post-translational modifications to fold
- Amino-terminal and carb**
Many antibiotics used clinically are inhibitors of protein synthesis. Discuss why some antibiotics are specifically used in the treatment of bacterial infections while others are used to treat yeast and fungi.
Yeast and fungi are eukaryotic while bacteria are prokaryotic. As such, many of the antibiotics that are harmful to bacterial cells are relatively harmless to eukaryotes.
What is a signal sequence and what is its function
An amino acid sequence, often at the amino terminus, that signals the cellular fate or destination of a newy synthesized protein
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