Pick one of the papers listed at http://www.nature.com/collections/vbqgtrPrepare a 10’-15’ journal club about it for March 16

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrPrepare a 10’-15’ journal club about it for March 16•Spend ~ 5’ setting the stage: • what is the general question?• Why is it important?• What was previously known?• What were the outstanding questions?

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrPrepare a 10’-15’ journal club about it for March 16•Spend ~ 5’ setting the stage: • what is the general question?• Why is it important?• What was previously known?• What were the outstanding questions?

•Then state the specific question addressed in your paper

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrPrepare a 10’-15’ journal club about it for March 16•State the specific question addressed in your paper•Next explain how they studied it• General overview of techniques first, then specifics• What were they trying to do?• how did they do it?

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrPrepare a 10’-15’ journal club about it for March 16•Then state the specific question addressed in your paper•Next explain how they studied it• General overview of techniques first, then specifics• What were they trying to do?• how did they do it?

•Then describe their results

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrPrepare a 10’-15’ journal club about it for March 16•First state the specific question addressed in your paper•Next explain how they studied it•Then describe their results• General overview first

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrPrepare a 10’-15’ journal club about it for March 16•First state the specific question addressed in your paper•Next explain how they studied it•Then describe their results• General overview first• Then specific experiments

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrDescribe their results• General overview first• Then specific experiments• Specific purpose of each experiment• How they tested it• Data they collected• Controls!!

• How they analyzed it

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrDescribe their results• General overview first• Then specific experiments• Specific purpose of each experiment• How they tested it• Data they collected• Controls!!

• How they analyzed it• Conclusions they drew

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrDescribe their results• General overview first• Then specific experiments• Specific purpose of each experiment• How they tested it• Data they collected• Controls!!

• How they analyzed it• Conclusions they drew• Your interpretation• Do you agree?• How could they improve?

Pick one of the papers listed at http://www.nature.com/collections/vbqgtrDescribe their results• General overview first• Then specific experiments• Specific purpose of each experiment• How they tested it• Data they collected• Controls!!

• How they analyzed it• Conclusions they drew• Your interpretation• Do you agree?• How could they improve?

Translation initiation mediated by RNA looping

PNAS 112: 1041-1046 (Jan 27, 2015)

Basic question: How do euk ribosomes find start codon?

Basic question: How do euk ribosomes find start codon?•Current model = ribosomal scanning: euk assemble 43S complex at 5’ cap and scan down to first AUG

Basic question: How do euk ribosomes find start codon?•Current model = ribosomal scanning: euk assemble 43S complex at 5’ cap and scan down to first AUG•Why don’t 40% of mRNA start at first AUG?

Basic question: How do euk ribosomes find start codon?•Current model = ribosomal scanning: euk assemble 43S complex at 5’ cap and scan down to first AUG•Why don’t 40% of mRNA start at first AUG?•How do IRES (internal ribosome entry sites) work?• Found in many viral mRNAs eg polio, picornavirus

Basic question: How do euk ribosomes find start codon?•Current model = ribosomal scanning: euk assemble 43S complex at 5’ cap and scan down to first AUG•Why don’t 40% of mRNA start at first AUG?•How do IRES (internal ribosome entry sites) work?• Found in many viral mRNAs eg polio, picornavirus

•Authors propose RNA-looping finds initiator AUG

Basic question: How do euk ribosomes find start codon?Authors propose RNA-looping finds initiator AUG •proteins which bind near initiator AUG guide initiation complex

Basic question: How do euk ribosomes find start codon?Authors propose RNA-looping finds initiator AUG •proteins which bind near initiator AUG guide initiation complex•Tested by making constructs containing internal RNA-binding protein sites and measuring effect on translation

Authors propose RNA-looping finds initiator AUG •proteins which bind near initiator AUG guide initiation complex•Tested by making constructs containing internal RNA-binding protein sites and measuring effect on translation•Made a fusion between eIF4G and the MS2-RNAbp, and then added MS2 binding sites at various places

• Made a fusion between eIF4G and the MS2-RNAbp, and then added MS2 binding sites at various places

• eIF4G was mutated so it can’t bind eIF4E & PABP, yet it still associated with eIF3 & the 40S subunit

• Made a fusion between eIF4G and the MS2-RNAbp, and then added MS2 binding sites at various places

• eIF4G was mutated so it can’t bind eIF4E & PABP, yet it still associated with eIF3 & the 40S subunit

• Capped the mRNA with A to ensure no eIF4E binding

Authors propose RNA-looping finds initiator AUG •Made a fusion between eIF4G and the MS2-RNAbp, and then added MS2 binding sites at various places w/in mRNA encoding luciferase•Assayed by transfection into HEK293 cells, then measuring firefly luciferase/Renilla luciferase activity 4 hours later

• Assayed by transfection into HEK293 cells, then measuring firefly luciferase/Renilla luciferase activity

• No activity w/o eIF4G& MS2 binding site• Stem-loop (whichblocks back-tracking)had no effect

• Assayed by transfection into HEK293 cells, then measuring firefly luciferase/Renilla luciferase activity

• No activity w/o eIF4G& MS2 binding site• Stem-loop (whichblocks back-tracking)had no effect

• Assayed by transfection into HEK293 cells, then measuring firefly luciferase/Renilla luciferase activity

• No activity w/o eIF4G& MS2 binding site• Stem-loop (whichblocks back-tracking)had no effect• Concluded that eIF4Gbound to the mRNA wassufficient to guide 40S tothe start codon

Next tested effect of the eIF4G binding on a downstream ORF •Put Renilla luciferase & firefly Luciferase CDS on same mRNA

• Put Renilla luciferase & firefly Luciferase CDS on same mRNA

• FLuc depended on eIF4G binding• G-capped RLuc did not, but A-capped RLuc did

Put Renilla & firefly Luciferase CDS on same mRNA•Observed same effect with the encephalomyocarditis virus (EMCV) IRES

Put Renilla & firefly Luciferase CDS on same mRNA•Observed same effect with the encephalomyocarditis virus (EMCV) IRES•Overall conclusion: RNA binding proteins that can interact with the 40S complex allow it to find the start codon by looping the RNA

PROTEIN TARGETINGAll proteins are made with an “address” which determines their final cellular location

Addresses are motifs within proteins

PROTEIN TARGETINGAll proteins are made with “addresses” which determine their locationAddresses are motifs within proteins Remain in cytoplasm unless contain information sending it elsewhereTargeting sequences are both necessary & sufficient to send reporter proteins to new compartments.

2 Pathways in E.coli http://www.membranetransport.org/1.Tat: for periplasmic redox proteins & thylakoid lumen!• Post-translational

2.Sec pathway• Co-translational

Sec pathway part deux•SRP binds preprotein as it emerges from rib & stops translation•Guides rib to FtsY•FtsY & SecA guide it to SecYEG , where it resumes translation & inserts protein into membrane as it is made

Periplasmic proteins with the correct signals (exposed after cleaving signal peptide) are exported by XcpQ system

PROTEIN TARGETINGProtein synthesis always begins on free ribosomes in cytoplasm

2 Protein Targeting pathwaysProtein synthesis always begins on free ribosomes in cytoplasm1) proteins of plastids, mitochondria, peroxisomes and nuclei are imported post-translationally

2 Protein Targeting pathwaysProtein synthesis always begins on free ribosomes in cytoplasm1) proteins of plastids, mitochondria, peroxisomes and nuclei are imported post-translationallymade in cytoplasm, then imported when complete

2 Protein Targeting pathwaysProtein synthesis always begins on free ribosomes in cytoplasm1) Post -translational: proteins of plastids, mitochondria, peroxisomes and nuclei 2) Endomembrane system proteins are imported co-translationally

2 Protein Targeting pathways1) Post -translational2) Co-translational: Endomembrane system proteins are imported co-translationallyinserted in RER as they are made

2 pathways for Protein Targeting1) Post -translational2) Co-translational: Endomembrane system proteins are imported co-translationallyinserted in RER as they are madetransported to final destination in vesicles

SIGNAL HYPOTHESISProtein synthesis always begins on free ribosomes in cytoplasmin vivo always see mix of free and attached ribosomes

SIGNAL HYPOTHESISProtein synthesis begins on free ribosomes in cytoplasmendomembrane proteins have "signal sequence"that directs them to RER

Signal sequence

SIGNAL HYPOTHESISProtein synthesis begins on free ribosomes in cytoplasmendomembrane proteins have "signal sequence"that directs them to RER“attached” ribosomes are tethered to RER by the signal sequence

SIGNAL HYPOTHESIS• Protein synthesis begins on free ribosomes in cytoplasm• Endomembrane proteins have "signal sequence"that directs them to RER• SRP (Signal Recognition Peptide) binds signal sequence when it pops out of ribosome & swaps GDP for GTP

SIGNAL HYPOTHESISSRP (Signal Recognition Peptide) binds signal sequence when it pops out of ribosome & swaps GDP for GTP•1 RNA & 7 proteins

SIGNAL HYPOTHESISSRP binds signal sequence when it pops out of ribosome

SRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER

SIGNAL HYPOTHESISSRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER Ribosome binds Translocon & secretes protein through it as it is made

SIGNAL HYPOTHESISSRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER Ribosome binds Translocon & secretes protein through it as it is madeBiP (a chaperone) helps the protein fold in the lumen

SIGNAL HYPOTHESISRibosome binds Translocon & secretes protein through it as it is madesecretion must be cotranslational

Subsequent eventsSimplest case:

1) signal is cleaved within lumen by signal peptidase2) BiP helps protein fold correctly3) protein is soluble inside lumen

Subsequent eventsComplications: proteins embedded in membranes

proteins embedded in membranesprotein has a stop-transfer sequence

too hydrophobic to enter aqueous lumen

proteins embedded in membranesprotein has a stop-transfer sequence

too hydrophobic to enter aqueous lumentherefore gets stuck in membraneribosome releases translocon, finishes job in cytoplasm

More ComplicationsSome proteins have multiple trans-membrane domains (e.g. G-protein-linked receptors)

More ComplicationsExplanation: combinations of stop-transfer and internal signals-> results in weaving the protein into the membrane

Sorting proteins made on RERSimplest case: no sorting• proteins in RER lumen are secreted

Sorting proteins made on RERSimplest case: no sorting• proteins in RER lumen are secreted• embedded proteins go to plasma membrane

Sorting proteins made on RER

Redirection requires extra information:

Sorting proteins made on RER

Redirection requires extra information:

1) specific motif

2) receptors

Sorting proteins made on RER ER lumen proteins have KDEL (Lys-Asp-Glu-Leu) motif Receptor in Golgi binds & returns these proteinsER membrane proteinshave KKXX motif

Sorting proteins made on RERGolgi membrane proteins • cis- or medial- golgi proteins are marked by sequences in the membrane-spanning domain

• trans-golgi proteins have a tyrosine-rich sequence in their cytoplasmic C-terminus

Sorting proteins made on RERPlant vacuolar proteins are zymogens (proenzymes)

signal

signal

VTS

VTS

Barley aleurain

Barley lectin

mature protein

mature protein

Sorting proteins made on RERPlant vacuolar proteins are zymogens (proenzymes), cleaved to mature form on arrival• targeting motif may beat either end of protein

signal

signal

VTS

VTS

Barley aleurain

Barley lectin

mature protein

mature protein

Sorting proteins made on RERlysosomal proteins are targeted by mannose 6-phosphate M 6-P receptors in trans-Golgi direct protein to lysosomes (via endosomes)M 6-P is added in Golgi by enzyme that recognizes lysosomal motif