Protein Synthesis (Kieft L1)

Question 0

Essential components of the activation step?

Answer 0

20 aa.'s
20 aminoacyl-tRNA synthetases
32 or more tRNA's
ATP
MG2+

Question 1

Steps in protein synthesis?

Answer 1

1. Activation of aa.’s
2. Initiation
3. Elongation
4. Termination & ribosome recycling
5. Folding and posttranslational processing

Question 2

Essential components of the Initiation step?

Answer 2

mRNA
N-Formylmethionyl-tRNA(fMet)
Initiation codon in mRNA (AUG)
30S/50S (bact.) 40S/60S (euk) ribosomal subunits
Initiation factors (IF 1, IF2, IF3)
GTP
MG2+

Question 3

Essential components of Elongation step?

Answer 3

Functional 70S or 80S ribosome (initiation complex)
Aminoacyl-tRNA specified by codons
Elongation Factors (EF-Tu, EF-Ts, EF-G)
GTP
Mg2+

Question 4

Essential components of Termination and Ribosome recycling?

Answer 4

Termination codon (UAA, UAG, UGA)
Release factors (RF-1, RF-2, RF-3, RRF)
EF-G
IF-3

Question 5

Essential components of Folding and posttranslational processing?

Answer 5

Specific enzymes, cofactors, other components for removal of initiating residues & signal sequences

Add’l proteolytic processing
Modification of terminal residues and attachment of acetyl, phosphoryl, methyl, carboxyl, carbohydrate or prosthetic groups

Question 6

Where does attachment of aa. to tRNA occur?

Answer 6

in cytosol (not on ribosome)

Question 7

What is aminoacylation of tRNA?

Answer 7

attaching proper aa. to proper tRNA using ATP as energy and Mg2+ for stabilization.

once attached the rRNA is “charged”

Question 8

What must be true of the carboxyl group on the incoming aa. for the new peptide bond to be formed?

Answer 8

The carboxyl group must be activated (phosphate taken from ATP is attached to the carboxyl C) to initiate bond formation

Question 9

Basic steps in initiation.

Answer 9

1. mRNA binds smaller rib. subunit (30S/40S)
2. larger rib. subunit binds (50S/60S)
   - –> this forms the initiation complex (70S/80S)
3. initial tRNA base pairs with start codon (AUG, Met)
4. GTP is used for energy
5. cytosolic initiation factors support the process

Question 10

Basic steps in Elongation

Answer 10

1. New polypeptide lengthened by att. of successive aa’s.
2. new aa’s carried to rib. by tRNA
3. new aa’s bp with corresp. codon
4. elongation factors assist.
5. GTP hydrolysis provides e. for tRNA att. and mvmt of pp. along rib.

Question 11

Basic termination steps.

Answer 11

1. Termination codon in mRNA signals end (UAA, UAG, UGA)
2. New pp. released from ribosome
3. Release Factors assist.
4. Rib. recycled for another round

Question 12

Basic steps of posttranslational processing and folding

Answer 12

1. PP fold to proper 3D form
2. Enzymatic processing of pp. (before or after folding)
   
   • addn of acetyl, posphoryl, methyl, carboxyl
   • att. oligosaccharides or prosthetic groups

Question 13

Ribosome is made up of what components?

Answer 13

Bacteria: 30S, 50S; 5S, 23S rRNA - note, there is no protein within 18 Å of the active site
final rib. complex = 70S

Eukaryotic: 40S, 60S; rRNA as well
final rib. complex = 80S

Question 14

What should we really call a ribosome and why?

Answer 14

Ribozyme

most of it is rRNA material
rRNA does the enzymatic work

Question 15

The mRNA passes ______ the two _______ _________ during translation.

Answer 15

between the two ribosomal subunits

Question 16

What are the three sites of the ribosomes that bind tRNA’s?

Answer 16

A = aminoacyl site
P = peptidyl site
E = exit site

Question 17

What binds to the aminoacyl site?

Answer 17

aminoacyl-tRNA’s

Question 18

What binds to the peptidyl site?

Answer 18

aminoacyl-tRNA

Question 19

What binds to the exit site?

Answer 19

uncharged tRNA after it has completed its task on the ribosome

Question 20

What makes up a tRNA?

Answer 20

single strand of RNA folded into 3D structure

Question 21

How does a tRNA pick up an aa?

Answer 21

Each tRNA recognizes exactly one aa.
aa. attaches to the amino acid arm
Aminoacyl-tRNA synthetases esterify the aa. to its site on the tRNA

Question 22

Where does the tRNA pick up its aa?

Answer 22

in the cytosol (not on the ribosome)

Question 23

What is the “shape” of a tRNA?

Answer 23

in 2D it is a 4-arm cloverleaf.
*aa. arm *anticodon arm *D arm *TpsiC arm (longer have 5th arm)

in 3D is a “twisted L” or gun form

Question 24

What does the small subunit on the ribosome do/contain?

Answer 24

Contains decoding groove *mRNA's pass through decoding groove *tRNA's enter and read the message in the decoding groove

Question 25

What does the larger ribosomal subunit contain?

Answer 25

Catalytic Center!!! *PTC = peptidyl transferase center *is made of RNA --> ribozyme !! uses RNA to perform catalysis

Question 26

Where is the amino acid arm of the tRNA?

Answer 26

at the 3' end

Question 27

What is special about the D arm?

Answer 27

contains the unusual nucleotide dihydrouridine (D)

Question 28

What bond does the aminoacyl-tRNA synthesize?

Answer 28

an ester bond R-C(=O)-OR' between aa and its tRNA

Question 29

How many tRNA's can carry a particular aa?

Answer 29

usually one, sometimes more if the aa has multiple codons

Question 30

How many aa's can a tRNA carry?

Answer 30

one

Question 31

how many aa's and tRNA's can an aminoacyl-tRNA synthetase work with?

Answer 31

one aa and one or more tRNA's (when the aa has multiple codons)

Question 32

What is the function of the tRNA anticodon loop?

Answer 32

base pairs with the mRNA message codon in the usual complimentary base pairing way ---> wobble pairing at the 3rd codon allows some tRNA's to pair with more than one codon

Question 33

What do initiation factors do?

Answer 33

bring the ribosome to the message and assist in assembling the translation machinery

Question 34

How many initiation factors do bacteria have? How many initiation factors do eukaryotes have?

Answer 34

Bact: 3 IF1, IF2, IF3 Euk: over a dozen...

Question 35

What do elongation factors do?

Answer 35

Deliver the tRNA's and move the ribosome down the message

Question 36

What do termination / recycling factors do?

Answer 36

end translation at a stop codon (UAA, UAG, UGA) dissociate the subunits for reuse

Question 37

What is the reading frame?

Answer 37

the start codon AUG sets the reading frame (codon = 3 nucleotides long). there is no overlap from codon to codon

Question 38

When/where is the reading frame established?

Answer 38

during initiation when the machinery is being established at the start codon (AUG, Met)

Question 39

To begin initiation, the _________ and ________ must assemble in the ________ of the ribosome.

Answer 39

start codon (5'AUG) Met-tRNA P-site

Question 40

The next aa. in the growing chain arrives at the __________ before it enters the ________ site.

Answer 40

A site | P site

Question 41

What is the direction of the synthesis of the growing pp chain?

Answer 41

N ---> C

Question 42

What is the residue at the Amino Terminal of every new pp?

Answer 42

Met (5'AUG)

Question 43

How many tRNA's does bacteria have for Methionine?

Answer 43

Two. One is used for the 5'AUG start codon (fMet). One is used for an internal AUG methionine code.

Question 44

Methionine side chain?

Answer 44

-CH2-CH2-SH-CH3

Question 45

How many tRNA's does eukaryotic cells have for Methionine?

Answer 45

One regular tRNA(Met)

Question 46

During elongation, where does the incoming tRNA bind?

Answer 46

First to the A site, then to the P and E sites. | only the first tRNA(Met) binds directly to the P site

Question 47

The GTP that provides the energy for the initiating pp. bond is attached to _________.

Answer 47

IF2

Question 48

In eukaryotes ________ is required to bind the 7-methylguanosine cap at the _________ end of the __________.

Answer 48

eIF 4E 5' mRNA

Question 49

What other eIF's are assembled during euk. initiation?

Answer 49

4G, 4A, 4B, etc.

Question 50

In eukaryotes, how does the ribosome find the start codon?

Answer 50

scans to find AUG (Met)

Question 51

What are IRES's?

Answer 51

Internal ribosomal entry sites. often exploited by viruses after shutting down the host cell cap-dependent synthesis

Question 52

What step is considered to be the most controlled/regulated in translation?

Answer 52

initiation

Question 53

During elongation, what delivers the inconing aa-tRNA to the A site?

Answer 53

Euk: EF1A Bact: EF-Tu

Question 54

How does termination begin?

Answer 54

Recycling Factor reads stop codon (UAA, UAG or UGA) in the A-site RF moves to tRNA's space and terminates peptide chain

Question 55

How does termination end?

Answer 55

Release of pp chain dissociation of subunits protein goes off to fold, receive modifications

Question 56

What molecule is the decoding molecule?

Answer 56

tRNA

Question 57

How many possible codons are possible?

Answer 57

4^3 = 64 yay, math!

Question 58

What is degeneracy in regards to codons?

Answer 58

multiple 3-nucleotide codons for a single aa.

Question 59

How many aa's are specified by just the first two nucleotides in the codon?

Answer 59

6 aa. where just the first two nucleotides matter.

Question 60

In general, are charged tRNA's involved in regulation of translation?

Answer 60

No. There are generally enough of the around.

Question 61

If the wrong aa. is put on a tRNA, how is this "proofread"?

Answer 61

1. The aminoacyl-tRNA synthetase has a built-in proofreading mechanism that we don't understand very well. (they are diff. for diff. synthetases) 2. The elongation factor that brings the charged tRNA to the rib. won't bind very well.

Question 62

Where did the names for the ribosomal subunits come from? 30S / 50S / 70S 40S / 60S / 80S

Answer 62

sedimentation rates through a centrifuge

Question 63

What happens at the A site?

Answer 63

Charged tRNA's enter into the ribosome

Question 64

What happens at the P site?

Answer 64

tRNA gives up its aa to the growing pp chain.

Question 65

What happens at the E site?

Answer 65

uncharged tRNA slides here then exits

Question 66

During bacterial initiation of translation, first _________ and ________ bind to the _________ subunit.

Answer 66

IF1 and IF3 | 30S

Question 67

Once IF1 and IF3 are bound to 30S, _________ binds to ________ using ________ as a guide.

Answer 67

mRNA 30S Shine-Dalgaro

Question 68

Once mRNA is bound to the 30S subunit at initiation, _________ brings a special ________ to the ________ to pair with the _______codon.

Answer 68

IF2 fMet-tRNA (bacteria only) P-site AUG

Question 69

Hydrolysis of ________ bound to ________ provides energy to release initiation factors and binding of ___________.

Answer 69

GTP IF2 50S subunit

Question 70

What is special about the Shine-Delgardo sequence?

Answer 70

is how ribosome recognizes the correct AUG start codon. is upstream form AUG Watson-Crick base pairs with 3' end of rRNA within small subunit will position AUG correctly in the P-site

Question 71

During elongation, peptide bond formation is catalyzed by _______ in the __________.

Answer 71

Peptidyl Transferase in the | Peptidyl Transferase Center (PTC)

Question 72

In eukaryotes, scanning for AUG start codon, scanning is _________ dependent and is more ________.

Answer 72

ATP | regulated

Question 73

In eukaryotes, what elongation factor ________ brings charged ________ to the vacant _______ site.

Answer 73

EF1 A tRNA's A

Question 74

After a peptide bond is formed, _______ and _______ hydrolysis triggers movement of the _________ and ________ one codon in the 3' direction.

Answer 74

EF2 GTP mRNA and tRNA

Question 75

Where does the energy for peptide bond formation come from?

Answer 75

The ATP used in tRNA charging.

Question 76

Formation of one peptide bond requires _________ high energy bonds

Answer 76

4

Question 77

What are the high energy bonds used to form a peptide bond?

Answer 77

1. Charge tRNA: 2 (ATP ----> AMP) 2. Deliver aa-tRNA to A site: 1 (GTP ---> GDP) 3. Translocation: 1 (GTP ---> GDP)