Proteins are chains of amino acids joined together
There are 20 different amino acids (A.A.)
Features of proteins:
Amino group – has N
Carboxyl group – has C
R group – “side chain”, each amino acid has a different R
The bond that joins two amino acids together is called a ____ ____
peptide bond
Proteins are also called ____
polypeptides
N-terminus
front of polypeptide, similar to 5’ on DNA/RNA
C-terminus
back end of polypeptide, similar to 3’ on DNA/RNA
tRNA structure
Each tRNA has 4 to 5 “arms”
Anticodon arm – contains the anticodon, interacts with mRNA sequence
Amino acid arm – attaches to amino acid
Other arms – structural, interact with ribosome, tRNA synthetase
tRNATyr
tRNA that recognizes a codon for Tyrosine, but does not necessarily have a Tyrosine amino acid attached to it.
Tyr-tRNATyr
tRNA that recognizes a codon for Tyrosine, and is “charged” with a Tyrosine amino acid.
Aminoacyl-tRNA
a tRNA with an amino acid attached to it.
Base modification
specific nucleotides are modified
Cleavage
the ends of the transcript are removed
CCA addition
a CCA is attached to the 3’ end of the transcript. This is what the amino acid attaches to
Introns removed only in....
eukaryotes
At which location, A, B, C or D, will the
anticodon be found?
D. )
At which location, A, B, C or D, will the
CCA attached?
D. )
tRNA Activation
Each tRNA must have its correct amino acid attached to it
2 steps: 1) Adenylylation and 2) tRNA charging
Both steps catalyzed by aminoacyl-tRNA synthetases (AATS), within the same active site
There are 20 different aminoacyl-tRNA synthetases, 1 for each amino acid
There are more than 20 tRNAs though, so each AATS can recognize more than one tRNA
tRNA Activation, Step 1: Adenylylation
Amino acid + ATP converted into aminoacyl-AMP + PPi
AMP attaches to carboxyl group of amino acid
Pyrophosphate (PPi) is generated in this reaction, which is later hydrolyzed into 2 phosphates, providing the energy to drive this reaction as well as make it irreversible
tRNA Activation, Step 2: tRNA charging
aminoacyl-AMP + tRNA into aminoacyl-tRNA + AMP
Aminoacyl transferred off of AMP onto the tRNA’s CAA arm
2 classes of aminoacyl-tRNA
synthetases do this differently
Class I - attach amino acid onto 2’OH of CCA
Class II – attach amino acid onto 3’OH of CCA
The addition of the charged amino acid to the
3’ adenine of a tRNA is being carried out by
a. Class I aminoacyl-tRNA synthetase.
b. Class II aminoacyl-tRNA synthetase.
B.)
Necessary
Something is necessary for a function when you need it to carry out that function
How to test if item A is necessary for a function
Remove item A and see if function is retained
Sufficient
Something is sufficient for a function when you can get function with only that thing
How to test if item A is sufficient for a function
Add item A in isolation (where you only have item A) and see if function can occur
You identify another motif (motif K) which contains a helix-turn-helix motif,
and you’re very sure it is the DNA binding motif for protein X. Which
strategy would best show that the motif is sufficient for DNA binding?
A. Create a fusion protein with motif K, plus the transactivation domain of
another protein and see if the fusion protein activates transcription of a
plasmid that contains the regulatory site for protein X
B. Create a nonsense mutation in the motif and see if it can still bind DNA
C. Delete that motif from the gene and see if the mutant protein can bind
to DNA
D. Create a mutant protein X that cannot bind DNA, then try to create a
reversion mutation to restore DNA binding. See if the mutation and
reversion mutation occur within the motif
A.)
Aminoacyl-tRNA synthetases can distinguish between tRNAs
The identity nucleotides are the
nucleotides of a tRNA that are recognized by their specific synthetases (orange circles).
Identity nucleotides are scattered throughout the tRNA
Each synthetase recognizes a unique set of identity nucleotides
Two different tRNAs for the same amino acid will share the same identity nucleotides, which allows them to be recognized by the same synthetase.
AA-tRNA proofreading
Case 1 – A larger AA trying to attach to a smaller AA’s tRNA
Amino acid binding site
If AA too big – doesn’t fit
If AA is too small - fits
AA-tRNA proofreading
Case 2 – A small AA trying to attach to a larger AA’s tRNA
Ile-tRNA synthetase has an acylation site, as well as a separate proofreading site
Bacteria:
Initiator Met tRNA
tRNAfMet
Bacteria
Internal Met tRNA
tRNAMet
________ converts Methionine to N-formylmethionine (fMet)
transformylase
fMet
Formyl group attaches to N-terminus of fMet, preventing fMet from being able to attach to an AA in front of it. fMet can only be the first AA.
Cannot be added internally
tRNAfMet is the only tRNA that is recognized by the ribosome initiation complex
Eukaryotes:
Initiator tRNA
tRNAiMet
Eukaryotes:
Internal tRNA
tRNAMet
In both bacteria and eukaryotes, ______ often remove the Nterminal Met, so many mature proteins don’t have Met as the first AA.
aminopeptidases
Ribosomes contain 2 subunits (Bacteria)
Small subunit - 30S
rRNA – 16S
Protein – 21 total subunits (S1 to S21)
Function – mRNA, tRNA assembly
Large subunit – 50S
rRNA – 5S and 23S
Protein – 36 total subunits (L1 to L36)
Function – catalyze peptide bond formation
Ribosomes contain 2 subunits (Eukaryotes)
Eukaryotes
Similar to bacteria but slightly
more complex, more proteins
40S small and 60S large
Puromycin
mimics tRNA, but binds directly to the large subunit, ribosomes will attach it to a growing polypeptide chain
New amino acids cannot be attached to puromycin, so it terminates translation and is therefore a powerful antibiotic
In this experiment, puromycin is just the substrate for forming peptide bonds
Ribozyme
RNA with enzymatic activity
The ribosome has 3 tRNA binding sites (name them)
A site
P site
E site
A site
– Acceptor site – where new tRNAs enter ribosome
P site
– Polypeptide site – where growing polypeptide chain is held
E site
– Exit site – where tRNAs are expelled after amino acid removal
Which ribosomal subunit holds the mRNA?
D. )
Which ribosomal subunit catalyzes peptide bond formation?
D. )
Shine-Dalgarno sequence
consensus sequence in front of start codon
Recruits small subunit to mRNA
Directs mRNA start site to correct position on ribosome
Base pairs with 16S rRNA on small subunit
Bacterial proteins involved in initiation
IF-1 – Fills A site to prevent tRNAs from binding
IF-2 – escorts initiator tRNA
IF-3 – prevents large subunit from binding
Translation Initiation in Bacteria, Step 1a-blocking
IF-1 and IF-3 bind to 30S small
subunit
IF-1 – blocks A site, prevents tRNA binding
IF-3 – blocks large subunit from binding
Translation Initiation in Bacteria, Step 1b- mRNA recruitment
mRNA attaches to 30S small subunit
uses Shine-Dalgarno sequence to position start codon right at P site
Translation Initiation in Bacteria, Step 2 recruitment of initiator tRNA
IF-2 binds to initiator tRNA
IF-2 is bound to GTP
Has GTP-hydrolase activity
Initiator tRNA (fMet-tRNAfMet) binds to start codon
rRNA binds to unique sequence on initiator tRNA (reason why internal tRNAMet doesn’t bind)
tRNAfMet can only bind to the P site
No other tRNAs can bind to P site
Translation Initiation in Bacteria,
Step 3 – recruitment of large subunit
30S changes conformation to kick out IF-3, allowing 50S large subunit to bind
Hydrolysis of GTP to GDP causes IF-1 and IF-2 to leave
Initiation complex completed
Both large and small subunits bound
mRNA with start codon lined up
Initiator tRNA bound to start site in P site of ribosome
What is/are the function(s) of the Shine-Dalgarno sequence?
E. )
Which factor is responsible for preventing tRNAs from prematurely base
pairing with the mRNA codons?
D. )
What does the hydrolysis of GTP to GDP help to facilitate?
D. )
Initiation in Eukaryotes, Step 1– blocking sites on small subunit
eIF1A binds to and blocks A site on small subunit to prevent tRNA binding
eIF3 (and eIF1) block large subunit from assembling
Initiation in Eukaryotes, Step 2 – loading initiator tRNA
eIF2 binds to initiator tRNA (Met-tRNAi Met) and escorts into P site of small subunit.
eIF2 is also bound to GTP
unique sequences on initiator tRNA allow eIF2 to bind
Initiation in Eukaryotes, Step 3 - loading the mRNA
eIF4F – complex that binds to 5’ cap of mRNA and escorts it to small subunit. (no real bacterial equivalent, but partly serves similar role as Shine-Dalgarno sequence)
Contains 3 factors
1) eIF4E – Binds to 5’ cap
2) eIF4A – ATPase and RNA helicase
3) eIF4G – Adapter, binds to eIF3 and eIF4E, linking mRNA to small subunit
Binding of eIF4F/mRNA to pre-initiation complex requires hydrolysis of ATP (by eIF4A)
Unlike bacteria, mRNA binds at 5’ cap (not start codon)
Initiation in Eukaryotes, Step 4 – Scanning for the start codon
Scanning – complex travels along mRNA until first start codon is found, then stops
Kozak sequence helps identify start codon (similar to Shine-Dalgarno)
Scanning implies that translation usually starts with the first AUG from the 5’ cap
Initiation in Eukaryotes, Step 5 – Loading large subunit
Both GTPs (bound to eIF2 and eIF5B) are hydrolyzed This drives the release of all initiation factors
Unlike bacteria, 2 GTPs are required (in bacteria, only 1)
Plus, 1 ATP was required to load mRNA
eIF4F and 5’ cap of mRNA also released
Large subunit can now bind to pre initiation complex and assembly of the initiation complex is now complete
Like the Shine-Dalgarno sequence in bacteria, the eukaryotic Kozak
sequence helps the small subunit to find the start codon. What other
eukaryotic factor helps to fulfill a different part of the function of the
Shine-Dalgarno sequence?
D. )
Which factor is not bound to GTP or ATP, or involved in its hydrolysis?
A. )
What is a feature of eukaryotic translation initiation that is different from bacteria?
A. )
Bacterial Polysomes
Multiple ribosomes can translate the same mRNA simultaneously
In bacteria, this can even happen while the mRNA is still being transcribed
Multiple RNA polymerases can also be transcribing the same DNA at once
Eukaryote Polysomes
eIF4G (part of eIF4F complex) can bind to poly-A binding protein (PABP)
Connects 5’ cap to poly-A tail, forming a circle
Facilitates translational regulation
Internal Ribosomal Entry Site (IRES)
Most of eukaryotic translation is called cap-dependent translation, but translation can also happen without using the 5’ cap and first start codon
Internal ribosomal entry site (IRES) – sequence that can bind eIF4F and direct ribosome assembly and translation away from the 5’ cap
Used by viruses that block cap dependent translation, but allows their own genes to translate
Viruses cleave eIF4G to block its ability to tether mRNA to small subunit
Small eIF4G fragment sufficient to direct IRES to ribosome
Some eukaryotic genes also use IRES
IRES is also used by molecular biologists to express two genes off the same transcript
Essentially makes eukaryotic polycistronic transcripts
What does eIF4G do?
A. Tethers the mRNA to the small subunit
B. Tethers the 5’ cap of mRNA to the poly (A) tail
C. Hydrolyzes GTP to separate eIF4F from the small subunit
D. Hydrolyzes ATP to bind the mRNA to the small subunit
E. A and B
A and B
A virus shuts down cap-dependent translation, which of these happen next?
