trinucleotide sequence that codes for an amino acid
Crick predicts existence of an _____ ____ that reads DNA (or mRNA) sequences and “translates” to amino acid sequence
adapter molecule
Transfer RNA connects _____ and _____
mRNA; protein
anticodon
triplet nucleotide sequence on tRNA that base pairs with codon on mRNA
The ____ position of the anticodon binds to the ____ position of the
codon, and vice versa
first; third
Anticodons will be listed _' to _'
3; 5
If the codon on the
mRNA is GCA (5’ to 3’),
what is its anticodon?
C. )
There are only ___ amino acids for ___ amino acid-encoding codons (+ __ stop codons)
20; 61; 3
The Genetic Code is ________
degenerate
degenerate
multiple codons can encode the same amino acid. Consequence of there being 64 possible codons but only 20 amino acids
Codon family
when 4 codons specify the same amino acid
Start Codon
AUG- Met (methionine)
Stop codons (3)
UAA, UAG, UGA
no tRNAs that recognize stop codons
Genetic code is read in .....
non-overlapping triplets (3 nucleotides at a time)
an mRNA has __# of reading frames depending on which nucleotide you start at
3
A double stranded DNA can potentially be transcribed in either direction leading to __# of potential of reading frames
6
open reading frame
A sequence that has a start codon, then a long stretch of codons, and then a stop codon all in the same reading frame
not a question just a tip...
How do you tell if a DNA or RNA sequence contains an open reading
frame?
1) Does it have a start codon? (AUG)
2) Does it have a stop codon? (UAA, UAG, UGA)
3) Are the start and stop codons in the same reading frame?
Inosine can pair with _, _, or _
C, U, A
Adenosine in 1st anticodon position
of tRNA is converted to _____
inosine (I)
A minimum of ___ tRNAs can potentially decode all codons
32
Humans have ___ different tRNAs
47
There are many copies of each tRNA
Why?
To ensure survival if any of them are mutated Also ensures there’s enough of each tRNA being transcribed
The 61 codons can potentially be recognized by ___ different tRNAs, due to wobble
32
In humans, there are __ tRNA genes, and most anticodons are shared by several copies of the same basic tRNA gene
415
Codon Bias
Some amino acids prefer particular codons, offering additional ways to regulate translation
Mutations happen to ___ (not ____), but affect the _____ sequence
DNA; RNA; mRNA
Single base substitutions: (define and give the 3 types)
Change of a single base in the DNA sequence of a gene
1.) Silent
2.) Missense
3.) Nonsense
Silent
change in codon that does not change the amino acid sequence.
Example: GAA (Glu) mutated to GAG (Glu). Both encode Glu so the mutation is silent.
Missense
change in codon that results in a different amino acid encoded
Example: GAA (Glu) mutated to GAC (Asp). Changes Glu to Asp.
Some missense mutations are worse than others. Glu and Asp are both negatively charged and their substitution likely wouldn’t have a huge impact on the protein (unless in the catalytic site)
Nonsense
change in codon that creates an early stop codon
Example: GAA (Glu) mutated to TAA (UAA). Causes change from Glu to stop codon. Leads to a truncated protein that is often non-functional
Transition mutation
A purine is substituted for another purine. A to G or G to A. Most common type of mutation. Can lead to all 3 types of substitutions.
Frameshift mutants
insertion or deletion of nucleotides that alter the reading frame of the coding sequence
Most frameshift mutations result in .....
premature stop codon
Deletion mutants
deletion of one or more nucleotides. Sometimes, large blocks of genetic material are missing.
If the number of nucleotides inserted or deleted is the same, then the
reading frame is ______
restored
reversion mutation
A reversion mutation is where a mutation that is deleterious to the function of the encoded protein acquires a second mutation that allows the protein to function again
Examples of reversion mutation
1) A missense mutation (Lys to Gly) in the active site causes the protein to be non functional. A second mutation returns the amino acid to Lysine.
2) Same situation as above, but a second mutation changes the Gly to Arg, which is similar to Lys and the protein is mostly functional.
3) A nonsense mutation (Tyr to STOP) creates a truncated protein that is non functional. A second mutation changes STOP back into Tyr.
4) Same situation as above, but the second mutation changes STOP to Ser, allowing a full length protein and restores function.
5) A frameshift mutation is caused by the insertion of a single nucleotide. A second mutation causes a deletion of a single nucleotide near the site of the insertion. Protein function is restored.
in order for a reversion mutation to be identified, protein function must be .....
restored (fully or partially)
What causes single base substitutions?
Chemical carcinogens and many, many other things.
What causes frame shift mutations?
Intercalating agents insert themselves into the DNA double helix
Cause stretching of the DNA
During DNA replication, as the DNA polymerase passes through strands stretched by intercalating agents, they can cause insertion or deletion mutations
Acrididin Orange is an intercalating agent used to make frameshift mutations in DNA
Ionizing Radiation
a type of radiation that causes the release of electrons from molecules, ionizing them.
Can directly damage DNA
Can indirectly damage DNA by creating free radicals from water molecules, which damage DNA
Leads to double strand DNA breaks
During repair, these breaks can lead to large deletion mutations of several nucleotides in length
X-rays are a common form of ionizing radiation used to create mutations
loss of large chunk of sequence (ionizing radiation)
Overview: Transition mutation
A to G or G to A
Overview: Reversion mutations
second mutation that restores protein function
Crick and Brenner: T4 Bacteriophage
mutants
Define.... T4 Bacetriophage, Plaque Assay, B gene, Acridine, and the actual experiment
T4 Bacteriophage – virus that infects bacteria
Plaque assay – when mixed with bacteria and plated onto agar dishes, the plate will be confluent with growing bacteria, but not where bacteriophage is growing. The plaques are the dark regions with dead bacteria.
B gene – required for bacteriophage to infect multiple strains of E. coli. If mutated, will only grow on one strain.
Acridine – intercalating agent, low doses introduce single insertion/deletion mutations
Experiment – mutate B gene, try to grow on two E. coli strains. Non-functional B gene will allow it to only grow on one strain.
Crick and Brenner: T4 reversion mutants
Two mutant strains of Bacteriophage could be crossed and recombine their genetic material. If one strain had a single insertion, and the other strain a single deletion, then combined the two frameshift mutations would offset each other, and the normal reading frame would be restored. Could classify single mutants into two groups by whether they could complement one another: “+” group and “–” group.
Two plus mutants won’t complement one another, and two minus mutants won’t complement one another, but one plus and one minus will complement one another.
First evidence that code is read
in triplets
Crick and Leslie Barnett
Recombined three “plus” mutant strains
3 insertion (or deletion) mutations will restore reading frame
Why was it important for Crick to use a mutagen that created insertion/
deletions?
A) Single base substitutions would not answer the question about gaps in the
code
B) Single base substitutions cannot undergo reversion mutations
C) Unlike 3 insertions mutations, 3 single base substitutions would not
necessarily restore the protein function when recombined.
D) Single base substitutions would not affect the B gene’s functions
C.)
What tools do you need to decipher the genetic code?
1) Cell free in vitro translation system
Add ribosomes, tRNA, mRNA and produce polypeptides
2) mRNA of defined sequence
Needed to know the sequences of the mRNA you were adding
How do you stitch individual nucleotides together to get fully defined sequences?
3) Method to determine which polypeptides were produced
Needed to know which amino acids were generated
A “cell free” system for synthesizing protein
Steps: (5)
Polynucleotide phosphorylase – enzyme that normally degrades RNA, but if given excess NDP, will catalyze the reverse reaction and start attaching them together
They couldn’t control the sequence, but if they added only UDP, could create a polymer of poly (U) RNA (i.e. UUUUUUUUUU)
Created 4 different polynucleotides:
• Poly (U) – UUUUUUUUU
• Poly (A) – AAAAAAAAAA
• Poly (C) – CCCCCCCCC
• Poly (G) – GGGGGGGG
Severo Ochoa
mixed ADP and CDP at 5:1 ratio, to produce triplets of different frequencies
Gobind Khorana
created dinucleotide, trinucleotide, and tetranucleotide repeats
Maxine Singer
Produced
trinucleotides of defined
sequence (not long polymers
of semi-random sequence)
Niremberg and Leder
1) Trinucleotides (not long random polynucleotides)
2) Ribosomes (cell extracts)
3) tRNA (with radioactive amino acids
attached)
Ran mixture over nitrocellulose filter
Filter traps ribosomes with trinucleotides and correctly matched tRNAs
unbound trinucleotides and tRNAs pass through
Could identify which amino acid/tRNA matched the trinucleotide based on radiolabel
Ribosome filter binding assay
A single trinucleotide would cause one and only one aminoacyl-tRNA to bind to the ribosome. 61 out of 64 possible codons could be decoded this way
