Lecture 26 Flashcards
How many amino acids are there?
20
each has a different radical group
Describe the structure of an amino acid.
a carbon bound to…
- H
- amino group
- carboxyl group
- radical group
type of bond joining the amino acids
peptide
what is the protein’s primary structure
the amino acid sequence
secondary structure of protein
the chain of amino acids folds on itself
- alpha helix
- beta pleated sheet
tertiary structure of protein
the secondary structure folds further
quaternary structure of protein
joining of multiple subunits
Proteins are produced during…
translation
triplet code
the idea that 3 nucleotides on mRNA are used to specify 1 amino acid
Why did early researchers devise the triplet code?
Knowing there were many amino acids, this is the only way the 4 bases could produce the enough amino acids
Francis Crick
- proposed that the code would be read in a nonoverlapping manner
Brenner and Crick
established that a codon consists of 3 non-overlapping nts
Nirenberg and Mattaei
- first to determine that a specific RNA sequence coded for a specific amino acid
Nonoverlapping
need a 3 letter codon to provide for all 20 amino acids
Nirenberg and Mattaei experiment
- used a cell tree extract to provide miscellaneous molecules needed for translation and added an RNA molecule that contained uracil and all 20 amino acids
- made different amino acids radioactive in 20 different tubes with 14C
- allowed protein to be produced and checked if radioactive
- since mRNA contained only Uracil, the codons would be UUU repeating over and over
- polyUUU only produced in phenylalanine
- repeated fr polyC, polyA, did not work with polG
determined coding sequence for 3 codons
Triplet Binding Assay
- Nirenberg and Leder
- used to deduce the rest of the genetic code
Triplet Binding Assay process
- charged tRNA, ribosome, and trinucleotide bind and do not go through filter
1. set up 20 reaction tubes for each possible codon with different radioactive amino acid with trinucleotide, ribosome, and charged tRNAs
2. allow binding
3. filter out unbound amino acids
4. check if radioactive
only had radioactivity on filter paper when the trinucleotide was the codon for that amino acid
encoded 61 of 64 codons using this technique
The code is…
- unambiguous
- degenerate
- containing stop/stop punctuation
- without internal punctuation
- almost universal
unambiguous
a specific codon always codes for a specific amino acid
degenerate
there is more than one codon that codes for the same amino acid
stop codons
UAAA
UAG
UGA
start codon
AUG
no internal punctuation
mRNA is read beginning with the start codon without skipping any bases
almost universal
the code is almost always the same
How many amino acids have exactly 1 codon?
2
met and trp
How many amino acids have exactly 4 codons?
5
What is the highest number of codons for one amino acid?
6
in leucine, arginine, serine
Is there a pattern to the degeneracy of the code?
yes
the first two bases are more important than the last
wobble hypothesis
due to wobble in the anticodon, a single tRNA can pair with more than one codon
inosine
facilitates the wobble hypothesis
inosine is a base in tRNA and when present is in the 1st anticodon position. It can pair with uracil, adenine, and cytosine
ex of inosine in the wobble
Codons: GGU, GGC, and GGA
CCI will pair with all three
The codons that can bind to a tRNA with inosine must…
all code for the same amino acid
ex cannot code for tyrosine as only UAU and UAC code tyrosine while UAA is a stop codon
wobble in the anticodon
- an anticodon containing guanine in the 5’ position binds with 2 different codons
ex. AGG may bind with both UCC and UCU
steps of translation
- charging the tRNA
- initiation
- elongation
- termination
charging the tRNA means…
joining the amino acid to the tRNA at the 3’ end
initiation means…
the ribosome assembles on the mRNA and translation starts
elongation means…
the continued addition of amino acids to the growing peptide chain
termination means…
the stop of translation and release of the protein
enzyme that conducts the reaction to charge the tRNA
aminoacyl synthetase
Describe charging the tRNA
- the specific aminoacyl synthetase binds with its specific amino acid in the presence of ATP
- with magnesium as a cofactor, the Ppi is released and AMP is joined to the carboxyl of the amino acid
- the specific tRNA enters and displaces AMP
- this releases the AMP and joins the carboxyl end of the amino acid to the 3’ end of the tRNA
- the charged tRNA is then released from the enzyme
describe initiation
building a ribosome
- shine-Dalgarno sequence on the mRNA base pairs to a complementary sequence in the 16S rRNA
- the charged tRNA binds so that the anticodons binds to the AUG start codon on the mRNA forming the 30S initiation complex
- the large subunit of the ribosome binds to form the 70S initiation complex
ribosome sites
there are 3 sites that can hold tRNA
- A or aminoacyl
- P or peptidyl
- E or exit
where is the tRNA in the ribosome
the P site
where is the start codon in the ribosome
the P site
Describe elongation
- the start codon is in the P site and the next codon in the A site
- a tRNA with the correct anticodon and amino acid comes to the A site
- the 23S rRNA in the large subunit acts as a ribozyme to form the peptide bond between the two amino acid
- immediately after the dipeptide sits in the A site
- translocation of the ribosome occurs setting the dipeptide on the P site and freeing the A site for the next tRNA
- this continues until a stop codon sits on the A site
peptidyl transferase activity
- conducted by the 23S rRNA of the large subunit
- forms hydrogen bond between the amino acid at the P and A sites on the ribosome
ribozyme
when an RNA molecule acts to conduct an enzymatic function
reaction occurring to form the dipeptide in elongation
carboxyl end of the exisiting peptide is joined to the amino end of the incoming amino acid
produced from amino to carboxyl ends
translocation of the ribosome requires…
elongation factor G and GTP
describe termination
- stop codon arrives at A site
- release factor binds to stop codon
- the protein is released from the P site
- GTP converts to GDP and all other componenet dissociate
polysomes
multiple ribosomes translating the same mRNA simultaneously
in prokaryotes, transcription and translation can occur simultaneously since DNA isn’t in nucleus
Differences in eukaryotic initiation of translation
- cap and poly a tail impact initiation
- elongation and termination are similar but with more and different EFs and TFs
- it is not polycistronic
Kozac sequence
- surrounds the start codon in eukaryotic mRNA
5’ - ACCAUGG - 3’
Role of cap in eukaryotic translation
- small subunit of ribosome, initiation factors, and charged initiator tRNA recognize and bind to the cap
- the initiation complex moves down the mRNA until it finds the first start codon
Role of poly A tail in eukaryotic translation
- it interacts with proteins that bind the 5’ cap to enhance ribosomal binding by forming closed loop structure
