Protein Synthesis (Kieft L1) Flashcards by T L

Essential components of the activation step?

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

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Steps in protein synthesis?

Activation of aa.’s
Initiation
Elongation
Termination & ribosome recycling
Folding and posttranslational processing

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Essential components of the Initiation step?

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+

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Essential components of Elongation step?

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

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Essential components of Termination and Ribosome recycling?

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

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Essential components of Folding and posttranslational processing?

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

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Where does attachment of aa. to tRNA occur?

in cytosol (not on ribosome)

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What is aminoacylation of tRNA?

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

once attached the rRNA is “charged”

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

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

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Basic steps in initiation.

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

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Basic steps in Elongation

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

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Basic termination steps.

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

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Basic steps of posttranslational processing and folding

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

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Ribosome is made up of what components?

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

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What should we really call a ribosome and why?

Ribozyme

most of it is rRNA material
rRNA does the enzymatic work

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The mRNA passes ______ the two _______ _________ during translation.

between the two ribosomal subunits

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

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

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What binds to the aminoacyl site?

aminoacyl-tRNA’s

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What binds to the peptidyl site?

aminoacyl-tRNA

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What binds to the exit site?

uncharged tRNA after it has completed its task on the ribosome

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What makes up a tRNA?

single strand of RNA folded into 3D structure

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How does a tRNA pick up an aa?

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

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Where does the tRNA pick up its aa?

in the cytosol (not on the ribosome)

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What is the “shape” of a tRNA?

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

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

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

What does the larger ribosomal subunit contain?

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

Where is the amino acid arm of the tRNA?

at the 3' end

What is special about the D arm?

contains the unusual nucleotide dihydrouridine (D)

What bond does the aminoacyl-tRNA synthesize?

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

How many tRNA's can carry a particular aa?

usually one, sometimes more if the aa has multiple codons

How many aa's can a tRNA carry?

one

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

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

What is the function of the tRNA anticodon loop?

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

What do initiation factors do?

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

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

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

What do elongation factors do?

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

What do termination / recycling factors do?

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

What is the reading frame?

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

When/where is the reading frame established?

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

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

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

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

A site | P site

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

N ---> C

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

Met (5'AUG)

How many tRNA's does bacteria have for Methionine?

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

Methionine side chain?

-CH2-CH2-SH-CH3

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

One regular tRNA(Met)

During elongation, where does the incoming tRNA bind?

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

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

IF2

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

eIF 4E 5' mRNA

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

4G, 4A, 4B, etc.

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

scans to find AUG (Met)

What are IRES's?

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

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

initiation

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

Euk: EF1A Bact: EF-Tu

How does termination begin?

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

How does termination end?

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

What molecule is the decoding molecule?

tRNA

How many possible codons are possible?

4^3 = 64 yay, math!

What is degeneracy in regards to codons?

multiple 3-nucleotide codons for a single aa.

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

6 aa. where just the first two nucleotides matter.

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

No. There are generally enough of the around.

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

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.

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

sedimentation rates through a centrifuge

What happens at the A site?

Charged tRNA's enter into the ribosome

What happens at the P site?

tRNA gives up its aa to the growing pp chain.

What happens at the E site?

uncharged tRNA slides here then exits

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

IF1 and IF3 | 30S

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

mRNA 30S Shine-Dalgaro

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

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

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

GTP IF2 50S subunit

What is special about the Shine-Delgardo sequence?

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

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

Peptidyl Transferase in the | Peptidyl Transferase Center (PTC)

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

ATP | regulated

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

EF1 A tRNA's A

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

EF2 GTP mRNA and tRNA

Where does the energy for peptide bond formation come from?

The ATP used in tRNA charging.

Formation of one peptide bond requires _________ high energy bonds

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

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