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Biochem 385 Unit 3 > Lecture 35: Protein Synthesis > Flashcards

Lecture 35: Protein Synthesis Flashcards

1
Q

size of bacteria ribosome

A

70S ribosome

50S and 30S subunits

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2
Q

eukarotic ribosome

A

80S ribosome

40S and 60S subunits

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3
Q

protein synth starts at

A

AUG codon (Met)

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4
Q

bacteria start site recognition

A

Shine Delgarno Sequence

complementary sequence to 16S rRNA before the AUG

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5
Q

eukaryotic start site recognition

A

scan for 5’ AUG

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6
Q

peptidyl transfer rxn

A

catalyzed by 23S (28S) rRNA which acts as a ribozyme

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7
Q

amino acid charged tRNA molec binding overview…

A

bind to A site
catalysis in P site
uncharged tRNA exits from E site

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8
Q

Why GTP hydrolysis??

A

to move the ribosome

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9
Q

what is a ribozyme

A

an enzyme made of RNA

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10
Q

what do ribosomes do?

A

protein synthesis!!!

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11
Q

Bacterial protein synth

A

happens at same time as RNA synth!!!

RNA pol and ribosomes work together
Many ribosomes can work at the same time?

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12
Q

eukaryotic protein synth

A

happens in CYTOSOL

MANY ribosomes translating the PROCESSED mRNA transcript at the same time (polyribosome complex)

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13
Q

Protein synth starts….

A

at 5’ end of mRNA with first amino acid in Nterminus of polypeptide

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14
Q

ribosome structure

A

large and small ribosomal subunits

contains 3 tRNA sites covering 3 codons in the mRNA

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15
Q

A site

A

aminocyl

entry site for charged tRNA

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16
Q

P site

A

peptidyl

contains catalytic residues in rRNA

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17
Q

E site

A

exit

where uncharged tRNA is expelled from ribosome

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18
Q

at what sites does base pairing between anticodon of tRNA and codon of mRNA take place??

A

A and P sites of ribosome ONLY

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19
Q

size of ribosomal subuints… molecular terms and Svedberg units

A

sedimentation rate

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20
Q

bacterial ribosome

A

several RNA molecs
35+ proteins
large subunit is 50S
total: 70S

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21
Q

eukaryotic ribosome

A

parge subunit is 60S
total 80S
35+ proteins

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22
Q

Why is the S value for the bacterial ribosome (70S) smaller than the sum of the S values for the 2 subunits (50S ad 30S)

A

its not simply additive
sedimentation is more than just mass
also takes shape into consideration when associated, the molec seems smaller and more compact than the 2 indiv sub units

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23
Q

what is the catalyst for the peptidyl trasferase rxn?

A

rRNA

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24
Q

does a ribosome without proteins work?

A

not well, but it does work a little

25
Q

one codon for Met

A

5’AUG3’

26
Q

Shine Dalgarno sequence

A

in bacterial mRNA
upstream of initiator Met
forms base pair contacts with 16S rRNA molec in small ribosomal subunit

purine rich
if it isn’t the Shine-Dalgarno doesnt match the 16S rRNA sequence, it will still bind, just with lower affinity

27
Q

Eukaroytic initiaition and AUG

A

NO Shine Dalgarno sequence

just can back and forth for the AUG closes to the 5’ end of the mRNA

28
Q

Formyl Methionine

A

in bacteria
initiator Met codon is recognized by special tRNA^fMet

first charged w/ Met by aminoacyl synthetase
then a.a. is formylated by rxn with transformulase to get initiator fMet-tRNA^fMet

29
Q

tRNA charged w/ formyl Met…

A

is only used at the initial Met codon

30
Q 
Met codons (AUG) within the open reading frame (not initial codon) uses what tRNA?
(bacteria)
A

tRNA^Met

NOT forymlated one

(byt still charged by same aminoacyl synthetase enzyme)

31
Q

overall bacterial protein synth initiation

step 1

A

formation of complex containing 30S ribosomal subunit and 2 initiator proteins (IF-1 and IF-3)
then bind to mRNA transcript at initiator codon and Shine Dalgarno seq

32
Q

overall bacterial protein synth initiation

step 2

A

complex recognized by GTP bound IF-2

facilitates binding of fMet-tRNA^fMet to AUG codon in P site of ribosome

33
Q

overall bacterial protein synth initiation

step 3

A

50S subunit binds and hydrolyzes GTP

release of all IF proteins

final product: 70S initiatior complex

34
Q

Eukaryotic initiation doesnt…

A

don’t use shine dalgarno or formyl Met

35
Q

Eukaryotic initiation

A

forms iniatior complex w. small ribosoma subunit (40S), eIF2, eIF4E, eIF4G, and PAB INCLUDES 5’ cap!, AUG codon, and PolyAtail at 3’ end

forms a LOOP

this gets recognized by large ribosomal subunit (60S)

scan for AUG start codon (5’ to 3’ direction)
start synth

36
Q

What mught be the advatage of using this type of “generic” protein complx for initation rather than a “Shine-Dalgarno” ribosome binding site in the 5’ end of the mRNA transcript?

A

more potential products!
another way of generating different products from the same RNA
you could miss the first AUG and star with the next one

37
Q

elongation in bacteria: Step 1

A

binding of GTP bound elongation factor
Ef-Tu faciliates DELIVERY of charged tRNA to A site after GTP hydrolysis

different because A site (not P)!!!

reload EF-Tu elong factor with another GTP to keep elongation going

38
Q

whats elongation

A

addition of next a.a. to nascent polypep chain

39
Q

when is the only time we use the P site

A

only binding of initator codon is centered on P site

40
Q

Step 2 of elongation

A

23S ribozyme catalyzes peptide bond formation
through rxn that is driven by proximity and orientation of substrates (2 a.a.s)

FORMATION OF PEPTIDE BOND dipeptide attached to second tRNA
conf change in tRNAs, but base pairing stays same

41
Q

Step 3 of elongation

A

conf change in ribosomal complex
binding of GTP bound elong factor (EF-G) (uses nrg of GTP hydrolysis)
translocate ribosome in 3’ direction

everything moved over one site
A is open, stuff is in T and P, ready for new stuff in A

42
Q

net effect of elongation steps

A

net effect: A site is now centered on NEXT codon
dipeptidyl-tRNA centered in P site
there is room for next incoming charged tRNA

43
Q

Where will the 3rd amino acid end up?

A

on the BOTTOM

amino acid terminus is formed first, we add to the BOTTOM

44
Q

Termination

A

one of the 3 termination codons appears in A site

GTP independent binding of terminator proteins (RELEASE FACTORS (RF)

45
Q

Release factors

A

promote hydrolysis and release of polypeptide

then they dissociate

46
Q

binding of EF-G after RF binding

A

binding of next EF-G and ribosmome recycling factor (RRF) causes dissociation of ribosome
IF-3 binds to 30S to get readu for another round

47
Q

dissaemblation of the ribosomal complex

A

as upstream ribosome on mRNA transcripts approchaes SAME termination codon

48
Q

electron micrograph slide 17

A

electron micrograph slide 17

49
Q

Beginning with uncharged tRNA^AA1 and free AA, how many ATP equivalents are required to synth and release bacterial polypeptide of 50 aas (assume 1GTP=1ATP)

A

200 ATP equivalents!

See slide 18!
6 steps

50
Q

but are there usually only 50 aas?

A

more lie 500…

2000 ATPs needed

51
Q

Up to 90% of all of the chem nrg used in a cell is dedicatd to proten synth, which means it needs to be TIGHTLY REGULATED. why do you expect that the majority of proten synth regulation is at the level of DNA transcription NOT RNA translation

A

if we don’t need the product, don’t even start the process at making RNA. its a waste!

52
Q

how lots of antibiotics work

A

inhibiting protein synthesis

targer prokaryotic ribosomes

53
Q

puromycin

A

looks like 3’ end of charged tRNA
can bind to A site of ribosomal complex
polypeptide chain attached to the antibiotic (by peptidyl transferase rxn)
result: abortibe complex formation

54
Q

tetracycline

A

binds to A site
blocks binding of actual charged tRNA molec
effect: stalling ribosome on mRNA trasncript

55
Q

chloramphenicol

A

blocks formation of peptide bond

56
Q

erytheromycin

A

prevents ribosomal translocation

57
Q

steptomycin

A

interferes with codon base pairing

58
Q

the first antibiotic produced for wide-spread clinical use was penicilin, what is its mode of action?

A

stops synthesis of cell walls in bacteria

not targeted toward ribosomes

Biochem 385 Unit 3 (11 decks)

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