Translation (II)

Question 1

What do analogous and homologous mean?

Answer 1

-Analogous = not genetically related change, changed due to similar lifestyles
-Homologous = evolve from common ancestor

Question 2

What are the major components of the ribosome in eukaryotes? What is the version of this in prokaryotes/bacteria? What is the full ribosome called?

Answer 2

-40S, analogous to 30S
-60S, analogous to 50S
-80S ribosome

Question 3

Is the ribosome conserved in bacteria and eukaryotes?

Answer 3

-Broadly conserved

Question 4

Eukaryotic initiation steps

Answer 4

-eIF3 and eIF1a bind the 40S (small subunit)
-eIF2 + GTP + Met-tRNAi^Met bind to the 40s subunit along with eIF1, resulting in 43s preinitiation complex
-eIF4F binds to the 5’ cap of mRNA and poly() binding protein (PABP)
-eIF4F/mRNA bind to the 43s preinitiation complex
-eIF5 joins, preinitation complex begins scanning for the first AUG
-Identification of the AUG results in formation of the 48s preinitiation complex
-eIF2 hydrolyzes GTP to GDP + Pi and all initiation factors are released
-eIF5B hydrolyzes GTP to allow 60s subunit to join
-See diagram pg 10 L19

Question 5

What are the major differences in eukaryotic initiation of translation and E. coli

Answer 5

-Eukaryotic ribosomes are larger with 4 rather than 3 rRNAs
-There is a distinct initiator tRNA (tRNAi^Met), but Met is never formylated
—Charged = Met-tRNAi^Met
-The initiating codon (AUG) is identified differently

Question 6

Major points of identifying initiating codon in eukaryotes

Answer 6

-43S pre-initiation complex is formed
—eIF2-GTP binds Met-tRNAi^Met and associates with the 40S
-eIF4F complex binds to the 5’ cap, and recruits the 43S pre-initiation complex
-Result complex scans for the 1st AUG within a Kozak sequence, which requires ATP and codon-anticodon recognition

Question 7

What is the Kozak sequence? Where is it?

Answer 7

-The Kozak consensus sequence is GCCRCCAUGG
-It contains the leading AUG, which is 50-70nt from the 5’ end and the 5’ cap
-Changes in the purine (R) and last G reduce translation efficiency by 10x
-Kozak sequence does not base pair with rRNA

Question 8

Cap-dependent initiation

Answer 8

-Majority of proteins are translated this way
-eIF4G binds to the poly(A) binding protein (PABP) at the 3’ end of the mRNA, circularizing the mRNA
-Facilitates the translational regulating of gene expression due to coordination of poly(A) tail and 5’ cap
-Cap dependent translation includes Kozak sequence and initiation

Question 9

Cap-independent initiation

Answer 9

-Require an internal ribosomal entry site (IRES)
-Most IRES sequences are from viruses, and are large RNA elements
-Useful for transcriptional fusions

Question 10

What is notable about eIF2?

Answer 10

Binds and hydrolyzes GTP

Question 11

What is notable about eIF4F complex?

Answer 11

Coordinates binding 5’ cap and poly(A) tail

Question 12

What is notable about the Kozak sequence?

Answer 12

-Used for appropriate selection of AUG
-Does not base pair with ribosome

Question 13

What is notable about AUG selection and scanning?

Answer 13

-ATP dependent
-First available with a Kozak sequence

Question 14

What is notable about mRNA during initiation?

Answer 14

-mRNA is circularized
-Permits cross talk between poly adenylation and translation initiation

Question 15

What is vital to the function of an antibiotic?

Answer 15

-It must selectively inhibit the growth of microorganisms

Question 16

What is important to consider when looking for a target for antibiotics?

Answer 16

-Molecules must bind to targets that are poorly conserved in humans
-Ex: cell wall synthesis inhibitors

Question 17

Why do so many antibiotics target translation

Answer 17

-Translation is a good target for killing bacteria
-Antibiotics often stabilize non-productive states within translation or even antagonize specific processes

Question 18

Are all antibiotics used in health/clinically?

Answer 18

-Some are only used in the lab

Question 19

Puromycin basics

Answer 19

-Product of Streptomyces bacterium
-Resembles the 3’ end of Tyr-tRNA
-Causes premature termination of protein synthesis
-Derivatives have been used in the lab for labeling newly synthesized proteins

Question 20

Puromycin mechanism

Answer 20

-Puromycin binds to ribosomal A site, which catalyzes a snomal transpeptidation reactio to form peptidyl-puromycin
-RIbosome cannot catalyze the next transpeptidation because puromycin’s “amino acid residue” is linked to its “tRNA” via an amide rather than an ester linkage
-Protein synthesis is therefore aborted and the peptidyl puromycin is released

Question 21

Diphtheria toxin basics

Answer 21

-Toxin is a single protein cleaved into two fragments
—B fragment binds the cell membrane to allow A fragment in
—A fragment modifies EF2
-EF2 modified by A is inactive
-One molecule of A can ribosylate all EF2 in the cell

Question 22

Diphtheria toxin mechanism

Answer 22

-EF2 modified by A is inactive
—A fragment transfers ADP ribose from NAD+ to histidine of eEF2
-Only happens in eukaryotes because eEF2 has a modified histidine residue: diphthamide

Question 23

Ricin basics

Answer 23

-Small protein from seeds of castor oil plant
-Heterodimeric protein bound by one disulfide linkage
—–A chain is catalytic
—–B chain allows for toxin entry into the cell
-One molecule of ricin can inactivate all ribosomes in the cell

Question 24

Ricin mechanism

Answer 24

-N-glycoside hydrolase that cleaves adenine from a particular adenosine in the 28S rRNA
—–The A is only in eukaryote rRNA
—–Prevents binding by elongation factors

Question 25

How is gene expression controlled in prokaryotes

Answer 25

-Generally controlled at level of transcription
-Due to mRNAs having short lifetimes (a few minutes) and translation being coupled to transcription

Question 26

Rhe TPP Riboswitch

Answer 26

-Biosynthesis of thiamine pyrophosphate (TPP) requires transcription of the thiM gene
-The 5’ untranslated region (UTR) of thiM is a TPP-sensing mRNA element (aka a riboswitch)
-Results in concentration of the metabolite (TP) regulating translation of proteins needed for synthesis
-Prokaryotes

Question 27

Rhe TPP Riboswitch absence and presence

Answer 27

-Absence of TPP: mRNA assumes a conformation that allows the ribosome to bind translatio initation site (Shine-Dalgarno)
-Presence of TPP: an alternative secondary structure masks the Shine-Dalgarno sequence

Question 28

Control of gene expression in eukaryotes

Answer 28

-mRNAs have long lifetimes
-Control gene expression by controlling translation
-Cells decrease global rate of translation in response to
-Much of this decrease in translation is through phosphorylation of eIF2

Question 29

What are some stresses that eukaryotic cells might decrease the global rate of translation in response to?

Answer 29

-Limited nutrients/amino acids
-Infection by viruses
-ER stress/unfolded proteins (sudden increase in temp)

Question 30

eIF2 importance in initiation

Answer 30

-eIF2 is an αβγ trimer, where GTP is required for function
-eIF2-GTP binds Met-tRNAi^Met and associates with 40S, results in 43S pre-initiation complex
-eIF2B is the GTP exchange factor (GEF)
—–eIF2B and eIF2α form a complex

Question 31

How is translation controlled through phosphorylation of eIF2α

Answer 31

-When cells are under stress different eIF2α kinases can phosphorylate eIF2α, which can be reversed by an eIF2 phosphatase
-Phosphorylation inhibits protein translation
-Phospho-eIF2α forms a much tighter complex with eIF2B than unphosphorylated eIF2α
-Phospho-eIF2α sequensters eIF2B, preventing regeneration of eIF2-GTP required for initation
-Kinases have a sensing domain and a kinase domain