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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
one codon for Met
5'AUG3'
26
Shine Dalgarno sequence
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
Eukaroytic initiaition and AUG
NO Shine Dalgarno sequence | just can back and forth for the AUG closes to the 5' end of the mRNA
28
Formyl Methionine
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
tRNA charged w/ formyl Met...
is only used at the initial Met codon
30
``` Met codons (AUG) within the open reading frame (not initial codon) uses what tRNA? (bacteria) ```
tRNA^Met NOT forymlated one (byt still charged by same aminoacyl synthetase enzyme)
31
overall bacterial protein synth initiation step 1
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
overall bacterial protein synth initiation step 2
complex recognized by GTP bound IF-2 facilitates binding of fMet-tRNA^fMet to AUG codon in P site of ribosome
33
overall bacterial protein synth initiation step 3
50S subunit binds and hydrolyzes GTP release of all IF proteins final product: 70S initiatior complex
34
Eukaryotic initiation doesnt...
don't use shine dalgarno or formyl Met
35
Eukaryotic initiation
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
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?
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
elongation in bacteria: Step 1
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
whats elongation
addition of next a.a. to nascent polypep chain
39
when is the only time we use the P site
only binding of initator codon is centered on P site
40
Step 2 of elongation
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
Step 3 of elongation
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
net effect of elongation steps
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
Where will the 3rd amino acid end up?
on the BOTTOM | amino acid terminus is formed first, we add to the BOTTOM
44
Termination
one of the 3 termination codons appears in A site | GTP independent binding of terminator proteins (RELEASE FACTORS (RF)
45
Release factors
promote hydrolysis and release of polypeptide | then they dissociate
46
binding of EF-G after RF binding
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
dissaemblation of the ribosomal complex
as upstream ribosome on mRNA transcripts approchaes SAME termination codon
48
electron micrograph slide 17
electron micrograph slide 17
49
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)
200 ATP equivalents! See slide 18! 6 steps
50
but are there usually only 50 aas?
more lie 500... 2000 ATPs needed
51
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
if we don't need the product, don't even start the process at making RNA. its a waste!
52
how lots of antibiotics work
inhibiting protein synthesis | targer prokaryotic ribosomes
53
puromycin
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
tetracycline
binds to A site blocks binding of actual charged tRNA molec effect: stalling ribosome on mRNA trasncript
55
chloramphenicol
blocks formation of peptide bond
56
erytheromycin
prevents ribosomal translocation
57
steptomycin
interferes with codon base pairing
58
the first antibiotic produced for wide-spread clinical use was penicilin, what is its mode of action?
stops synthesis of cell walls in bacteria | not targeted toward ribosomes

Biochem 385 Unit 3 (11 decks)

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