Protein Synthesis Flashcards
Protein is synthesized from the ___ terminal to the ____ terminal
amino; carboxyl
Each ribosome contains a ___ site and an ___ site
P (peptidyl); A (acceptor)
Processed mRNA enters the cytoplasm where it associates with a ___
ribosome
A ___ RNA molecule (bearing the amino acid coded for in the first codon) binds to this codon in the mRNA
transfer
Two AAs are joined by a ___ bond
peptide
___ factors help to terminate protein synthesis. For some proteins, membrane targeting is largely determined by the composition of the first twenty or so AAs in its amino terminal. This amino sequence where the protein will be targeted is termed the ___ ___
releasing; signal peptide
All tRNA molecules are folded into a ___ structure. This folding is accomplished by ____ bonding between complementary bases
cloverleaf; hydrogen
Every tRNA molecule studied possesses 4 distinctive regions. For the region listed, name its significance:
The 3’ end of the molecule
the AA binding site
note: terminates with the sequences C-C-A
Every tRNA molecule studied possesses 4 distinctive regions. For the region listed, name its significance:
The TC loop
this is where the tRNA binds to the ribosome
Every tRNA molecule studied possesses 4 distinctive regions. For the region listed, name its significance:
The D loop
this loop contains a high percentage of dihydrouridine residues (recognized by the enzyme tRNA synthetase)
Every tRNA molecule studied possesses 4 distinctive regions. For the region listed, name its significance: anticodon loop
this loop contains the anticodon which is complementary to the codon of the messenger RNA
What are the possible functions of the rare or minor bases present in tRNA?
a. to disallow base pairing at certain points in order to form the cloverleaf structure
b. to disallow unwanted base pairing with the mRNA
c. to protect the tRNA molecules from the action of ribonucleases
Although there are 20 AAs, the cells possess more than 20 types of tRNAs. Why?
there are several codons which specify a given AA
There are six codons for serine and several tRNA which recognize the various codons for serine (tRNAser). These tRNAs are called ____ because, although they recognize different codons, they all recognize serine
isoacceptors
Base pairing is antiparallel. tRNA runs __ –> ___ and mRNA runs ___ –> ___
3’ –> 5’; 5’ –> 3’
In regards to the wobble hypothesis, the third nucleotide of the codon is degenerate. Sometimes usual base pairing takes place between the ___ base of the codon and the ___ base of the anticodon
third; first
For the following anticodon nucleotides; give the possible base pairs it can make in the wobble position with the codon:
U
G
I (inosine)
U in anticodon recgonizes A or G in codon
G in anticodon recognizes C or U in codon
I in anticodon recognizes U, C, or A in codon
The inosine nucleotide is formed by ____ ____ after tRNA is formed on certain tRNAs
adenosine deaminase
Three dimensional (tertiary) structure of tRNA molecules has shown that their molecules are __-shpaed. The __ end of the molecule is at one end and the ____ loop is at the other end.
L; 3’; anticodon
Activation involves the coupling of an AA to its corresponding tRNA. These reactions are catalyzed by a group of enzymes termed what?
amino acyl tRNA synthetases
The ___ bond between the AA and the tRNA molecules is a high energy bond. The AA is bound to the 2’ or 3’ - ___ group of the ribose of the terminal adenylate residue of tRNA
ester; OH
Since the pyrophosphate formed during the reaction is hydrolyzed, the activation process requires the hydrolysis of two high energy ____ bonds. This is equivalent to the use of __ ATPs
phosphate; 2
The aminoacyl-tRNA synthetases are highly specific for both the AA and tRNA that they bind. It is imperative that they exhibit a high degree of ___, since it is the ____ of the tRNA molecule which is recognized at the ribosome
specificity; anticodon
The number of tRNA synthetases is ___.
20
A single synthetase is able to recognize all the tRNA molecules that can be linked to the same AA. True or false?
true
Error corrections of the amino acyl tRNA occurs when the tRNA is no longer bound to the synthetase. True or false?
false; occurs when the tRNA is bound to the synthetase
What is the role of the AA in recognizing the anticodon for that AA?
none
note: if an AA attached to the tRNA is chemically altered, the altered form will be incorporated into the protein
Both the prokaryotic and eukaryotic ribosomes consists of two subunits. Each subunit is composed of what?
proteins and rRNA
In regards to the basic process of initiation of protein synthesis, give the first step
specific proteins, called initiation factors, dissociate the 70S (or 80s eukaryotic) ribosome into its two parts: 50s and 30s (40s and 60s eukaryotic)
In regards to the basic process of initiation of protein synthesis, give the second step
tRNAfmet (formyl methionine) is the initiation codon for prokaryotes. This tRNA binds to IF2-GTP which is required for binding to the 30S subunit
In regards to the basic process of initiation of protein synthesis, give the third step
the big subunit 50S reassembles with the 30S and the tRNAfmet introduces the first AA, methionine, into the protein. It binds to the initiation codon (AUG) at the P site. IF2 is lost from the complex with GTP hydrolysis
Three initiation factors are involved for prokaryotic initiation, IF 1, 2, 3. IF 1 and 3 bind to the free ___ subunit
30S
note: mRNA also binds to the 30S ribosome subunit
Three initiation factors are involved for prokaryotic initiation, IF 1, 2, 3. IF 2 (GTP) binds to ___ which binds to AUG at the P site
tRNAfmet
IF 2 is lost as ___ is hydrolyzed and ___ ribosome is formed and IF 1 and 3 are lost
GTP; 70S
True or false? Eukaryotic protein synthesis initiation has more and different factors
true
In regards to eukaryotic protein synthesis, eIF-4E is an ___ ___ binding factor which is essential for lining up the ___ ___ on the ribosome
mRNA cap; starting codon
The reassembled ribosome possesses two sites at which tRNA with their attached amino acids hybridize through their anticodons to the mRNA codons. ___ is the peptide site. ___ is the acceptor site. ___ is the exit site for empty tRNA ejection
P; A; E
In both prokaryotes and eukaryotes, protein synthesis begins by positioning of the AUG codon in the ___ site of the ribosome with either tRNAfmet or tRNAmet bound to it. The mRNA is ____ starting at the AUG at the 5’ end of the mRNA
P; translated
In regards to recognition in prokaryotes, between the 5’ end of the mRNA and the initiation codon (AUG) is a sequence of nucleotides called the leader sequence. Within this sequence is the ___-___ sequence (AGGAG, consensus) which is located approximately ___ nucleotides 5’ orientation to the initiation codon (AUG)
Shine-Dalgarno; 10
What does the Sine-Dalgarno sequence in prokaryotes permit?
binding and proper orientation of mRNA with ribosome
note: this orientation specifically locates the AUG codon for initiation
In eukaryotes, the AUG nearest the 5’ cap is the ____ codon
initiating
In regards to eukaryotes, since there will be only one initiating codon per mRNA molecule, only one protein can be made per eukaryotic mRNA molecule. True or false?
true
note: mRNA is monocistronic
The relative efficiency of eukaryotic message translation is determined by ___ ____ sequence surrounding the AUG start site. This messages which are close in sequence to this sequence will translate efficiently
Kozac Consensus
There are three stages of elongation which operate in a cycle. What are they?
binding, peptide bond formation, and translocation
In regards to the binding stage in elongation, the aminoacyl tRNA possessing the anticodon to the codon in the A site forms a complex with the elongation factor ___-___ and ___. This complex then binds to the ____ such that the aminoacyl tRNA is positioned to the A site
EF-Tu; GTP; ribosome
In regards to the binding stage in elongation, The GTP of the EF-Tu-GTP complex is hydrolyzed and EF-Tu is released. True or false?
true
In regards to the peptide bond formation stage in elongation, the peptide bond is now formed by a nucleophilic attack of the alpha-amino group of the AA in the A site on the ester bond between the AA and tRNA in the P site. This transferase reaction is catalyzed by what? What provides the energy for synthesis?
the ribosomal RNA; the energy for synthesis is from the activated AA attached to the tRNA
The final step in elongation is translocation. This involves movement of the ribosome downstream relative to what?
the mRNA by one codon; i.e. moved toward the 3’-end of the mRNA
In regards to translocation, when the ribosome moves, there is a release of the empty tRNA that was present in the __ site and transfer of the peptide-bearing tRNA from the __ site to __ site.
P; A; P
note: this step requires EFG and GTP hydrolysis
In regards to translocation, The A site becomes vacant and the next AA charged tRNA enters and binds (by codon/anticodon match). This continues over and over, adding one AA at a time until the protein is made. True or false?
true
On average: how many EF-Tu-GTP amino acyl tRNAs are tried in the A site before the correct one matches?
10
note: elongation step occurs in a few milliseconds
Eventually, a termination codon in the A site is reached, which no acylated tRNA recognizes. Releasing factors RF1, RF2, and RF3 cause the release of what?
the polypeptide chain from the ribosome and the ribosome dissociates and is ready to bind another message
The releasing factors cause the peptidyl transferase activity to switch to a ____ activity to release the completed polypeptide
hydrolase
In regards to polycistronic mRNA in prokaryotes, if an initiating codon AUG is not too far from terminal codon, what happens?
the 70S ribosome will not dissociate but will form a new initiation complex which will lead to translation of the second protein - thus one mRNA can code for several proteins and each one might be translated
As the ribosome translates mRNA, the 5’ end of the mRNA becomes exposed and a second initiation complex can form. Thus more than one ribosome can be bound to a particular mRNA molecule at any particular time. The structure formed by these ribosomes and the mRNA being translated is termed a ____
polyribosome
The distance between ribosomes is about ___ nucleotides
100 - therefore, for a mRNA containing 1000 nucleotides could be translated simultaneously by 10 ribosomes
In regards to importance of energetics of protein synthesis, in E. coli, ___-___% of the total energy appears needed for protein synthesis
30-50%
In regards to energetics of protein synthesis (activation), synthesis of the AA tRNA uses the equivalent of 2 ATP. This is for each AA introduced into the protein. The energy of the tRNA-AA bond is used to drive ___ bond formation
peptide
In regards to energetics of protein synthesis (elongation and transcription), 2 GTP/AA: a GTP is hydrolyzed for every AA delivered to the __ site and another GTP for every ___
A; translocaiton
The energy expended from GTP hydrolysis is used for changing the conformation of the translation machine and not for forming new covalent bonds. So how many high energy bonds are broken for each peptide bond?
4
The initiation AA in eukaryotes is ____. Regulation of protein translation may occur in certain instances by ___ of eIF-2
methionine; phosphorylation
note: phosphorylation of eIF-2 inhibits translation
Eukaryotic cells require more initiation and elongation factors than prokaryotic cells. True or false?
true
Binding of an mRNA to the 40S ribosomal subunit requires what structure in eukaryotes?
5’ - cap
Eukaryotic mRNAs are ____ and prokaryotes are ____
hint: cistronic
monocistronic; polycistronic
Eukaryotic cells contain both free and membrane bound ribosomes; whereas prokaryotic cells contain only free ribosomes
true
Signal peptide hypothesis for eukaryotes?
the signal for a ribosome to attach to the rough ER resides in the first 15-30 AAs at the amino terminal - called the signal peptide
The SRP (signal recognition particle) binds to what?
signal peptide just as it produces from the ribosomal complex
The SRP blocks translation until what?
the ribosome has docked with the ER membrane
The SRP contains __ proteins and a ___ molecule as a central component
6; RNA
The SRP receptor is found only in the ER and binds SRP when SRP is part of ribosomal complex. Targeting of proteins to the ER is a ___ dependent process
GTP
note: GTP binding to SRP and SRP receptor is required for targeting and GTP hydrolysis is required for regeneration of free SRP and SRP receptor
The SRP receptor forms a gated channel which opens up when?
upon ribosome cocking and is closed upon ribosome dissociation
Ribophorin
ribosomal receptor binds to the ribosome
Translocation: the signal peptide enters the ER lumen; protein elongation continues; the signal peptide is removed by ___ ____. ___ is required to transport protein through membrane
signal peptidase; ATP
Proteins destined for secretion enter the ER lumen complexity and are transported to the ___ for modification by ____
golgi; glycosylation
Proteins destined for incorporation into membranes contain stop-tranfer sequences (hydrophobic). What do these sequences signal for?
signal a stop to the translocation of the growing peptide chain into the ER lumen. Amazingly, the SRP receptor opens sideways to allow stop-transfer sequences to enter the membrane of the ER
Mitochondria contain DNA, which is designated as mtDNA. It resembles ____ DNA
prokaryotic
mtDNA is known to code for how many mitochondrial polypeptides?
9
Mitochondrial protein synthesizing resembles the eukaryotic system. True or false?
false; prokaryotic
Transport of protein into the mitochondria: most of the mitochondrial proteins are coded for by nuclear DNA and the proteins synthesized in the ER. The N-terminal of proteins targeted for the mitochondria are post-transitionally imported into the mitochondria. True or false?
true
This eukaryotic inhibitor inhibits peptide bond formation
cycloheximide
This eukaryotic inhibitor catalyzes the reaction of NAD+ with a eukaryotic elongation factor, resulting in inhibition of translocation
Diphtheria toxin
This eukaryotic/prokaryotic inhibitor inhibits peptide bond formation and can inhibit mitochondrial protein synthesis at high levels
chloramphenicol
This eukaryotic/prokaryotic inhibitor causes premature termination by acting as an aminoacyl-tRNA analogue; the amino acid chain in the P site is transferred to the inhibitor rather than the A site
puromycin
This prokaryotic inhibitor inhibits the binding of aminoacyl tRNAs to the A site on the ribosome (inhibits elongation)
tetracylcine
This prokaryotic inhibitor binds to the 30S ribosomal subunit and causes dissociation of the translation process
streptomycin
This prokaryotic inhibitor binds to the 50S ribosomal subunit and inhibits translocation
erythromycin
