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Flashcards in Protein Synthesis Deck (53):
1

Structure of tRNA

75 bases
50% bases base paired
10% modified bases

Acceptor stem, T loop, anticodon loop, D loop, variable loop

2

D Loop

Interacts with the synthetase enzymes

3

Acceptor Stem

Site of amino acid charging by the synthetase enzymes

Always has conserved sequence: CCA

4

T Loop

Interacts with 5S rRNA, helps position tRNA in the ribosome

5

How are bacterial tRNAs synthesized?

-Multimeric: 2 or more tRNAs in a transcript, which are processed to monomeric precursors; may also be embedded in rRNA transcripts

-cleaved by RNases at 5' and 3' ends

-bases modified

-CCA added to 3' end by tRNA nucleotidyl transferase (if not present already)

6

Processing of prokaryotic tRNA transcripts (multimers)

1. 5' end cleavage by RNase
2. 3' end cleavage by RNase
3. Bases modified
4. CCA added

7

Eukaryotic

Made as monomeric precursors
Leader sequence on 5' end is cleaved off
There is an intron by the anticodon which masks it --> tRNA cannot be used until fully processed/modified

Removal of tRNA intron involves endonuclease cleavage and ATP-dependent ligation

8

Prokaryotic Ribosome

50S- 23S + 5S rRNA
30S- 16S rRNA

70S

9

Eukaryotic Ribosome

60S- 28S + 5.8S rRNA
40S- 18S rRNA

80S

10

Processing rRNA

Transcribed by Pol I- moderately repetitive sequences
Self splicing ribozymes (no need for snRNPs)
Pol II brings in 5S rRNA

11

Where in the cell do ribosomal subunits self assemble from rRNAs and ribosomal proteins?

Nucleolus

12

Wobble sequence

Base pairing between codon/anticodon in 5' and middle codon bases must be perfect

Some flexibility in 3' base of codon (5' anticodon)

13

What does bacterial protein synthesis require?

mRNA (read 5' --> 3')

Ribosomes (subunits assemble on the mRNA)

Protein factors for initiation elongation, and termination

Activated tRNAs: one or more for each amino acid

Unique tRNA for initiation (f-met-tRNA)

14

Which translational protein factors require GTP energy?

IF2-GTP: to bind Met-tRNA

EF-Tu-GTP: Delivery of aminoacyl tRNA to ribosomes
EF-G-GTP: Translocation factors
RF-3-GTP: Release of complete polypeptide chain


eiF2-GTP (eukaryotes): Helps bind Met-tRNA

15

What does it mean that tRNA are adaptor molecules?

Bind both amino acids and mRNA codons

16

Function of aminoacyl tRNA synthetases

Activation of amino acids prior to tRNA attachment

17

2 steps in Amino Acid activation

Amino acid + ATP ----> aminoacyl adenylate: synthetase + PPi

Aminoacyl adenylate: synthetase + tRNA ---> aminoacyl-tRNA: synthetase + AMP

SUM: AA + ATP + tRNA ---> AA-tRNA + AMP + PPi

1) both carried out by aminoacyl tRNA synthetase
2)Synthetase specific for a given amino acid (ie: alanine tRNA synthetase)

18

How does synthetase control accuracy?

1) Each synthetase must recognize R group on amino acid AND some part of the nucelotide sequence of the corresponding tRNA (either anticodon, specific sequence on acceptor stem, sequences somewhere else on the molecule)

2)Each synthetase has an activation site (for activation and attachment) and a hydrolytic site to correct errors

3) The aa-tRNA complex that leaves the synthetase will be used in protein synthesis with no further means of correcting errors

19

How many energy bonds cleaves during aa activation?

Two high energy bonds cleaved/ aa activated- protein synthesis requires energy

1) cleavage of ATP
2) subsequent hydrolysis of pyrophosphate

20

Initiation of protein synthesis in prokaryotes

1) Formyl group added to met. Complex of initiator tRNA bound to fmet and IF2-GTP

21

First Codon in Prokaryotes

AUG

22

How to locate first codon (P)

Shine Dalgarno sequence

23

First amino acid (P)

IF2-GTP

24

Initiation factor (P)

IF2-GTP

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Small subunit binding codon (P)

AUG

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Ribosome assembly codon (P)

AUG

27

Elongation (P)

EF-Tu-GTP, peptidyl transferase, EF-G-GTP cycle

28

Termination (P)

Stop codon/RF-GTP protein

29

Shine Dalgarno Sequence

purine rich sequence: base pairs with 3' end of 16S rRNA. Usually 7-10 bases upstream of AUG start codon

AUG start codon is not necessarily the 1st AUG, but the AUG closest to the Shine Dalgarno Sequence

30

Elongation: Repeat of 3 Steps (P)

1) Codon-specific binding of the aminoacyl-tRNA to the A site of the ribosome mediated by EF-Tu-GTP

31

Polyribosome

Complex of mRNA with multiple ribosomes called a polyribosome or polysome

Once the start codon is free, another round of initiation can begin --> many ribosomes can read mRNA at the same time

32

Translation + Transcription

mRNA is read in translation from 5' --> 3' and is synthesized from 5' --> 3'

Therefore can be translated while being synthesized

33

Termination of Protein synthesis in prokaryotes

1) Stop codons: UAA, UAG, UGA appears in the A site
2)Stop codon recognized by RF1 and RF2 and they bind near the site so nothing else can come in
3) RF3-GTP binds ribosome
4) Hydrolysis of GTP
5) Cleavage of Ester bond (peptidyl transferase)
6) Release of protein, tRNA, mRNA, ribosomal subunits

34

Tetracycline

Inhibitor of Protein Synthesis
Blocks binding of aminoacyl-tRNA to A-site of ribosome (prokaryotes only)

35

Chloramphenicol

Inhibitor of Protein Synthesis
Resembles peptide bond; inhibits pepitdyl transferase activity (prokaryotes only)

36

Initiation of Translation differences between Eukaryotes and Prokaryotes

-capped mRNA
-initiation factors: eIF for eukaryotic
-met-tRNA is not formylated
-Ribosomal subunit/met-initiator tRNA/factors scan along mRNA to find AUG start codon- no Shine Dalgarno

37

First Codon (E)

AUG

38

How is first codon located (E)

Kozak Sequence

39

First amino acid (E)

Met

40

Where does the small subunit bind to? (E)

At the cap

41

Where does the ribosome assemble? (E)

AUG codon

42

Where is termination? (E)

Stop Codon/ RF protein

43

eIF4F

eIF4A-helicase
eIF4B- ATPase
eIF4E- binds eIF3 (on 40S ribosomal subunit)

*cap binding proteins*

44

eIF4G

scaffold protein on the cap

45

Kozak Sequence

Contains the first AUG after the AUG at the cap

46

Eukaryotic Translation

1) 5' cap interacts with the cap binding complex (A,B,E)

2) Presence of the eIF4F facilitates binding of the small-subunit tRNA

3) eIF4E facilitates binding of small subunit eIF3 initiator tRNA-eIF2-GTP (binds eIF3)

4)eIF4A (helicase), eIF4B (ATPase) move ahead to unwind secondary structure

5)small subunit eIF3-initiator tRNA eIF2 GTP scans the mRNA w/ the 40S subunit to find the AUG initiation codon (in Kozak consensus)

6)eIF5 then causes release of these accessory factors, GTP mediated re-organization and joining of the large subunit

47

Loop structure on Eukaroytic mRNA

Facilitates ribosome recycling

Cap binding proteins interact with Poly A binding proteins

48

HCV vs HIV

HCV
-RNA virus- flavivirus
-Infects liver cells
-uses host ribosomes to synthesize viral proteins but not into host DNA

HIV
-RNA virus- retrovirus
-infects human immune cells
-uses host DNA and genetic material to replicate

49

Lifecycle of HCV

(+) RNA virus infects cells and conducts translation with RNA-dependent RNA polymerase

5A is a replication protein on cell ER
and 5B is a RNA-dependent RNA polymerase on cell ER

Using those proteins, create (-) RNA

Change back to (+) RNA

Now can infect other cells

50

Ledipasvir

inhibits NS5A- replication complex protein

51

Sofosbuvir

inhibits NS5B- RNA dependent RNA polymerase (

52

If HCV RNA (+) is not capped, how can it be translated by the host ribosomes?

HCV IRES
Internal ribosome entry sequence
IRES is recognized by 40S subunit and eIF3 complex some how

53

Ataluren-Facilitated Translation

Ataluren is a drug to help with CF mutation class 1 with shortened protein

Drug helps shift ribosome, tRNA-trp binds to stop codon --> still can get same length protein as opposed to before when the trp could not bind to stop codon