Lec6+7Translation Flashcards
How does tetracycline inhibit protein synthesis?
Binds to bacterial 30s ribosomal subunit and prevent attachment of aminoacyl-T RNAs to the acceptor site of the ribosome
After either 30 or 50s ribosomal subunit
What is the mechanism of tetracycline resistance that is related to ribosome function?
Tet (M,O,Q,S)= resistance genes that make a cytoplasmic protein that interact with ribosome making it insensitive to tetracycline
What is the binding site for a lot of proteins?
the phosphate on the CTD of RNA pol 2
to hop on the RNA as its being synthesized
What is a glob?
Globs is what we see in real life, RNA is covered with proteins
What is the nuclear export/pore receptor?
finds nuclear uses ATP to get mRNA out
T/F the only thing needed for mRNA to leave is the nuclear export receptor
FALSE, YOU ALSO NEED ATP
What do SR do?
Recruites splicesomes
What do hNRNP do?
block the spliceosome proteins
RNA poly 2 has ____ ____ where ____ ____ are looking for place to add cap and they end up generating ____ that will interact with this complex for initiating factors for _____
Phos CTD; cap enzymes; RNAs; ribosomes
MRNA-cap binding complex:
5’cap interacts
Nucelar pore complex
aqueous channel to cytoplasm
diffusion for small molecules only (less than 50K daltons)
Nuclear export receptor needed for macromolecules, including finished mRNAs
For nucelar pore complex, diffusion is for small or late molecules? Tell me the important number size (NEED TO KNOW)
SMALL
<50K daltons
For nucelar pore complex, diffusion is for small or late molecules? Tell me the important number size (NEED TO KNOW)
SMALL
<50K daltons
Where does the catalytic energy come from since we do not have a protein that is peptide bond synthetase?
catalytic energy comes from rRNA
What happens in the nucleolus
Nucleolous is what we assemble RNA with
Polypeptides
-Creates large and small subunits of ribosomes
-Enzymes involved in processing of rRNA
Assembly w/ snoRNA
-Telomerase assembling with its RNA
Nucleolus assembles with the rRNA genes
The big rRNA gene is the starting point for creating the nucleolus
what assembles with the nucleolus
rRNA genes
What is the starting point for creating nucleolus?
big rRNA
What four rRNAs are needed for the completed Rb
18S,5.8S, 28S from one gene (nucleolus forms from this gene cluster)
5S from another gene (this is IMPORTED INTO THE NUCLEOLUS
What rRNA is imported into the nucleolus
5S
What are the large subunit rRNAs
5.8S, 28S, 5S
What is the small subunit rRNA
18s
Remember: mRNA can be translated once or many times?
many times to make many proteins
what do rRNA with modification function as?
catalysis the peptide bond, facilities the tRNA codon interaction
rRNA are gene duplicates or gene differences? explain
gene duplicates
exact duplicates we can only transcribe them once to only make one ribosome from rRNA transcime
there is no splicing (IVS instead)
What is the universal genetic code
Codons represent three nucleotides
REDUNDANCY is expected because there are only 64 combo of 3 nucleotides but only 20 AA
3 stop codes so 64-3 = how many combination that encode AA
If we have 64 combo, how many combinations will we have that encode AA
64-3=61
What is reading Frame shift
If we shift the reading frame, we shift the meaning of the codon, you can also tune into a stop codon very quickly
Cancers contain a lot of Fram shift mutation and end up with a truncated protein
**translate different proteins
Where does the amino acid attached to on the tRNA
3’ on the tRNA
what is the codon on 3’ of tRNA
CCA
For the CCA is it an independent of dependent template
independent
What is on the 5’ on the tRNA
Anticodon on tRNA for the codon on mRNA to bind
Where is the anticodon located
tRNA
where is the codon located
mRNA
What needs to be recognized for AA to bind
Modification spread around the tRNA,enzymes adding the AA to the tRNA need to recognize the tertiary structure of tRNA created by modifications
There are how many amino acetyl transferase enzymes?
20
What do acetyl transferase enzymes do?
That will only charge tRNA with a specific AA and recognize a different tertiary structure
Humans have __ different anticodons (____) but there are __ codons (mRNA) that code for amino acids
48 different anticodons (tRNA) but there are 61 codons (mRnA) that code for amino acids
Does we have mismatch in anticodon and codons, what do we do?
Wobble (IMPERFECT W/C BP) and it gives an extra bond to stabilize–> tRNA need flexibility
tRBA redundancy
Is wobble imperfect or perfect W/C BP
imperfect
What is added for wobble
Inosine purine before R group added for A or G
Where is the inosine added for woddle
ANTICODON 5’
tRNA base modifications help with “wobble” and provide what?
binding sites for aminoacyl-tRNA-synthetases that charge the tRNAs with amino acids
What are other modification besides inosine (lower yield)
4-thiouridine
N,N dimethyl G
Dihyrdo U
What do aminoacyl-tRNA synthetases need and what drives the reaction?
ATP is needed, pyrophosphates drives the reaction
What is aminoacyl-tRNA-synthetases doing?
added the amino acid to the tRNA on the 3OH via A.A.’s carbonyl groupie so that way the amino group is available to attach the carboxyl group of the preceding tRNA
Is tRNA synthetase specific of nonspecific for a particular amino acid? give example
specific!!!!
tRNA synthetase (tryptophanyl tRNA synthetase) with the amino acid tryptophan
Which of the base allows for wobble hypothesis? A, B, C, D?
A!!
It is ALWAYS on the 5 prime end of an anticodon
If incorrect AA is added in the synthesizing site but fits in the EDITING SITE, what happens?
it will be DEGRADED
If incorrect AA is added in the synthesizing site but fits in the SYNTHESIS SITE, what happens?
nothing, it will go on its merry way
How does peptide bond formation work?
Amino group of new AA attacks the carboxyl group of the proceeding AA to form a peptide bond
Then the processing tRNA goes away
Does bacterial and eukaryotic ribosomes similar of very different
similar
What are 3 sites in ribosomes when translating? what do they ‘do’
A site(acceptor): a new tRNA coming in
P site(peptide): tRNA holding growing peptide
E site (exit): when the tRNA is leaving
How is the amino acids linking in this growing polypeptide chain
Carboxyl group (old amino acid that was already linked) linking with amino group of a new AA
Now how is the polypeptide chain growing ?
so we are GROWING FROM N to C TERMINAL
Peptide bond formation between the amino acids are catalyzed by what?
rRNA
Ribosome is indeed a ”Ribozyme”
The ribosome brings 10^-7 fold increase
Peptide bond formation between the amino acids are catalyzed by what?
rRNA
Ribosome is indeed a ”Ribozyme”
The ribosome brings 10^-7 fold increase
What does rRNA do ?
rRNA facilitates codon-anti-codon interaction and also facilitates hydrolysis of GTP bound to elongation factor.
rRNA can help stabilize the anticodon codon
Interaction with non standard w-c base pairing by adding
a additional bond
What protects rna from exonuclease
poly A tail by creating a circle that stabilizes the RNA with the initiation factor but will eventually fall apart
We need what to recognition mRNA?
initiation factor is important to the poly a tail binding proteins
Another IF binds the cap binding complex which is why the cap and cap binding complex is important
*** w/o cap= no recognition of mRNA
What is the specific cap binding protein that is recognized by small Rn subunit
eIF4E
What is used for moving along the mRNA when locking for AUG
ATP!!!
What is used for binding Met to the ribosome (used by IF)
GTP and once bound it can leave
What is the Elongation factor?
like a chaperone protein that probably has a mechanism of attraction for the ribosome
EF-Tu
What is needed to being AA to ribosome
GTP
Explain elongation
Once proper fitting of elongation factor there is a release of GTP and a sing phosphate
No GTP=no translate
T/F elongation and initiation factors are important for translation?
True
How does termination happen?
3 stop codons recognize specific proteins that block the binding of any tRNA
We need the release factor to bind to bring translation to an end
What do we need to bring translation to the end?
release factor to bind
What are the roles of rRNA in the ribosome ?
Forms overall structure
Form A,P, E sites
Stabilized codon anticodon base pairing
Catalyzes peptide bond formation
Where are mRNA translated?
On polyribosome structures
are mRNA and ribosomes making polypeptides a one to one ratio?
NO
you can have one mRNA with multiple ribosomes making multiple polypeptides= amp of mRNA
If we had a polypeptide release what would happen?
If we had a polypeptide release, we would have the n term but not c term because it hasn’t added yet
If we had a polypeptide release what would happen?
If we had a polypeptide release, we would have the n term but not c term because it hasn’t added yet
The circled part would have what terminal
would have the N term and NOT c term
What is present in the nascent polypeptides?
-N term
-C term
-Both
-neither
N term
Puromycin does what?
Puromycin only produces the n term frag when added, you never see the c term without the n term
Causes the premature release of polypeptide chain
Antibiotics interfere with what?
translation
What is tetracycline effect?
blocks binding of aminoacyl-tRNA to the A site of ribosome
What is steptomycin effect
prevents the transition from translation initiation to main elongation and also causes miscoding
what are chaperones
help with proper folding by stopping the growing polypeptide from folding until it is complete
If you have a protein you have isolated from a ribosome from a tetracycline resistant bacteria w/ a A mutation, what can you do?
you can map it out and see where the protein Is located (small or large subunit)
Why can’t the polypeptide unfold itself after if there is no chaperone
As the peptide grows its going to seek an energy minimum (folds) but now it will take too much energy to unfold
what is an example of a chaperone?
heat shock protein 70
what does heat shock protein 70 (hip 70) do?
Coats polypeptide when it needs to and prevents folding
Processed mRNA is transported out of the nucleus through a _________ that interacts with a specific “nuclear export receptor” attached to the mRNA
nuclear pore complex
___________position enzymes along the rRNA to modify bases. These modifications provide for more flexible Watson-Crick base pairing during intra-molecular base pairing and provide binding sites for ribosomal proteins.
SnoRNA
___________position enzymes along the rRNA to modify bases. These modifications provide for more flexible Watson-Crick base pairing during intra-molecular base pairing and provide binding sites for ribosomal proteins.
SnoRNA
The _____________ is the site of synthesis of the large rRNA and the site of ribosome subunit assembly
nucleolus
The nucleolus is also the site of assembly of some other proteins that require a polypeptide to be associated with an RNA molecule for final protein structure and function, for example,_________.
telomerase
rRNA genes (rDNA) have many ___________, to facilitate the rapid production of large amounts of rRNA needed for ribosome construction and protein synthesis.
repeats
rRNA genes (rDNA) have many ___________, to facilitate the rapid production of large amounts of rRNA needed for ribosome construction and protein synthesis.
repeats
Codons consist of __________nucleotides. There are 61 codons for the amino acids and _____________ termination codons. Because three nucleotides are needed for each codon, the mRNA has three potential reading frames for establishing the amino acid sequence of the encoded polypeptide.
3
3
Codons consist of __________nucleotides. There are 61 codons for the amino acids and _____________ termination codons. Because three nucleotides are needed for each codon, the mRNA has three potential reading frames for establishing the amino acid sequence of the encoded polypeptide.
3
3
____________ are loaded onto their cognate tRNAs at the 3’ end of the tRNA, which is always an adenine, by amino acyl tRNA synthetases, which are specific for the amino acids and their cognate tRNAs and which use ATP to facilitate the bond formation.
amino acids
The amino acyl tRNA synthetases include an editing function that will lead to __________of the “wrong amino acid-wrong tRNA” combination.
removal or hydrolysis
Base modifications in the tRNAs help amino acyl tRNA synthetases identify the correct tRNA to bond with the correct ____________.
amino acid
__________ base pairing and tRNA redundancy add flexibility to interpretation of the codons in the mRNA.
Wobble
Polypeptide synthesis occurs from the__________terminal to the COOH terminal of the polypeptide. The last amino acid added to the polypeptide has a free COOH group.
N
Polypeptide elongation at the ribosome is facilitated by _________ factors that use GTP, for energy, to insert the proper tRNA (anticodon) into a Watson-Crick base pairing arrangement with the proper codon.
elongation
Polypeptide elongation at the ribosome is facilitated by _________ factors that use GTP, for energy, to insert the proper tRNA (anticodon) into a Watson-Crick base pairing arrangement with the proper codon.
elongation
rRNA facilitates _______________ hydrogen bond formation. rRNA will also facilitate the hydrolysis of GTP in the above elongation factor function if the proper codon-anticodon pair have come together.
codon -anticodon
Ribosome assembly begins with the ___________bound to the small subunit, followed by mRNA and large subunit recruitment. The met-tRNA anticodon will eventually base pair with the AUG codon at the start of translation.
MET (methionine) tRNA
Energy is required in the form of ATP and GTP to assemble the ___________and to begin translation.
Complete ribosome
Translation is terminated when a specific protein recognizes a ________________, preventing any further tRNA association with the ribosome and release of the polypeptide, following hydrolysis of the COOH group from the tRNA.
stop codon
Most eukaryotic ___________ contain many ribosomes, representing structures called polysomes or polyribosomes.
mRNA
____________ often work by inhibiting translation, by affecting specific steps of translation.
antibiotics
Proteins must fold properly following translation or the polypeptide is degraded. Some proteins fold into their proper tertiary structure during synthesis. Other proteins require ____________ and ATP to fold properly.
chaperones
Dr. Blanck: if you delete 2 nucleotides would you stay in the original reading zone ?
If you delete 3 nucleotides would you stay in the original reading zone?
no
Yes because you deleted a whole amino acid
Dr. Blanck: Inosine is a precursor to what
adenine and guanine
Dr. Blanck:Which of the following nucleotides is particularly important to extend the versatility of tRNA
inosine
Dr. Blanck: inosine in tRNA is important for:
wobble hypothesis
Dr. Blanck: how would we count for 61 codons biochemically with only 20 a.a.:
- tRNA redundancy
-woddle
-both
-neither
Both
Dr. Blanck: which of the following is important for the amino-acetyl synthetase for recognizing the proper amino acid in tRNA?
-tRNA tertiary structure
-tRNA anticodon
-both
-neither
only tRNA tertiary structure
Dr. Blanck: which of the following is the substrate for RNA pol
triphosphate
Dr. Blanck: what types of molecules recognize the stop codon ?
-Release protein
-tRNAs anticodons
-both
-neither
Release protein
