Week 10 - Regulation of Translation

Question 1

what initiates translation?

Answer 1

ribosome through first AUG codon

Question 2

Bacterial translation

Answer 2

• transcription and translation are coupled
• polysomes begin assembly on elongating mRNA

Question 3

Eukaryotic translation

Answer 3

• U protein complex is the primary message and can be spliced
• RNAP2 transcribing the gene
• mature mRNA transported into cytoplasm where ribosome goes onto mRNA and actively translates it

Question 4

shine dalgarno sequence –

Answer 4

8 bases upstream from AUG seq.

Question 4

Where does regulation of translation in eukaryotes occur?

Answer 4

cytoplasm

Question 5

Molecules required for initiation of translation in bacteria

Answer 5

• 70s ribosome
• 30s ribosome and 16S RNA
• 50S ribosome –> not as important as 30 and 70s
• IF1 Initiation factor 1
• IF2
• IF3
• Initiation fMet-tRNAfmet (f-methionine tRNA)

Question 6

Bacterial translation

Answer 6

• ribosome falls off mRNA –> will dissociate into 30s and 50s subunits when IF1 and IF3 bind to 30s
• fMet-tRNAfmet, mRNA and IF2 will bind to create 30s preinitiation complex (AUG AND ANTI-CODON HAVE NOT ALIGNED ATP)
• Initiation tRNA interacting with AUG = 30s initiation complex
• IF1 and IF3 are ejected = 30S RNA
• GTP in IF2 is hydrolyzed such that IF2 dissociates
• 50S subunit binds to 30s = 70s initiation complex (ready and poised for elongation of polypeptide chain)

Question 7

what is important for placing mRNA onto ribosome and finding first AUG?

Answer 7

anti-SD and SD
- Watson and crick bp between these seq –> allows initiating tRNA to find first AUG

Question 8

How is rate of translation regulated in Bacteria?

Answer 8

** reduce rate of initiation**

1. inhibiting or enhancing the presentation of a ribosome BS
   
   • SD seq or similar seq + AUG allow ribosome to recognized this as an initiation site of translation
2. inhibiting the mRNA from entering the ribosome
   
   • mRNA can bind but not enter ribosome channels
   • 1st initiating contact RNA makes contact with AUG

Question 9

What 4 factors influence the formation of secondary RNA structures?

Answer 9

1. Binding of Proteins
2. Binding of metabolites
3. Binding of RNA
4. Temperature

Question 10

Stem loop (SL) secondary structures

Answer 10

• SD seq and AUG are hidden here such that no initiation complex form and ribosome cannot find SD or AUG

Question 11

Binding of Proteins

Answer 11

• proteins binds to its mRNA to stabilize
• SD and AUG are in SL therefore no available for ribosome to bind
• S15 binds to mRNA expressed from gene required fro synthesis of this ribosomal protein

+/- Feedback loop: as S15 protein increases, its own synthesis is inhibited

Question 12

when is S15 expressed?

Answer 12

When IPTG inhibits lac repressor form binding to lac operator

Question 13

when are there high beta-galactosidase levels expressed?

Answer 13

-IPTG and therefore - S15

• no S15 to inhibit beta-galac.

Question 14

What mutation would result in high expression in presence of IPTG?

Answer 14

• mutation in S15 such that structure is destabilized
• mutation in stem loop structure such that beta-gala. expression is high when +/- S15

Question 15

riboswitch

Answer 15

senses levels of metabolites

Question 16

TPP

Answer 16

thiamine pyrophosphate

Question 17

what does TPP do?

Answer 17

regulates the level of translation of mRNA that encodes genes important for thiamine biosynthesis

Question 18

what occurs to mRNA when little TPP?

Answer 18

mRNA will form secondary structure and expose SD and AUG to 30s ribosome –> where the initiation complex can form

Question 19

What occurs when TPP binds to mRNA?

Answer 19

• induces another secondary structure to high SD and AUG from ribosome
• SD and AUG in stem loop –> initiation is SUPRESSED

Question 20

what can metabolites influence?

Answer 20

expression of gene by binding to secondary RNA structure

Question 21

Positive riboswitch

Answer 21

metabolite exposes SD and AUG so ribosome can bind

Question 22

Negative riboswitch

Answer 22

another secondary structure induced to high AUG and SD in hairpin

Question 23

Hfq

Answer 23

RNA chaperone protein that stabilizes structures between 2 RNAs (RNA negative regulation)

Question 24

Hfq binds to ____ end of RNA

Answer 24

5’

Question 25

RNA positive regulation

Answer 25

SD and AUG hidden in stem loop, when Hfq binds, RNA stimulates translation of another mRNA to expose SD and AUG –> ribsosome can now bind to initate translation

Question 26

thermosensor

Answer 26

RNA structures at 5’ end of bacterial mRNAs that can cause different secondary structure to form
lower temp = SD and AUG expose
higher temp = SD and AUG placed in stem loop

Question 27

when RNAP has gone through 3’ UTR, translation is ____ because SD and AUG bp with ___, hiding them

Answer 27

suppressed, 5’ end

Question 28

5’ Cap and polyA tail

Answer 28

present in eukaryotic mRNA

Question 29

Eukaryotic first AUG rule

Answer 29

initiation complex (40s) binds to mRNA, scans mRNA for first AUG, once found initiation of translation is set up, packaging of mRNA/ activation binds to 43s pre initiation complex where it is attached to mRNA, 5’ to 3’ scanning for first AUG, hydrolysis of ELF2-bound GTP and Pi release = 48S initiation complex

Question 30

packaging of mRNA

Answer 30

pre-requisite of translation

Question 31

elF4F complex

Answer 31

must bind to 5’ cap
- forms structure on 5’ cap
- structure binds to PolyA BP which creates circle so 5’ and 3’ ends are close in proximity

Question 32

IRES (internal ribosome entry site)

Answer 32

-required for translation of internal cistrons
- RNA genome has multiple cistrons that need to be translated