Wk10 Transcription and translation extra practice questions answers PDF

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Course packet 11 answers - Dr Gorksi...

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MCDB1150(Course(Packet(week(10(transcription(and(translation( ! 1.! The!bacteria!E.#coli!has!a!4.6!million!base!pair!genome,!which!contains!an!estimated!4,330!protein! coding!genes.!You!are!studying!an!E.#coli!strain!that!initiates!transcription!inefficiently!and!randomly.! What!protein!is!likely!defective?!Sigma!factor! ! ! ! 2.! We!can!synthesize!DNA!in!vitro!(in!a!test!tube).!For!this!to!work,!we!need!to!start!with!a!template! doubleLstranded!DNA.!The!first!challenge!will!be!(as!written,!not!really!a!question).!But!we!know!that! we!will!need!to!denature/melt!open/break!the!H!bonds!between!the!double!stranded!DNA!(in!a!test! tube!we!usually!use!heat).!Next,!we’ll!need!something!to!“prime”!and!provide!a!3’OH,!such!as!an! RNA!primer.!We!will!need!a!DNA!polymerase!and!free!deoxyribonucleotide!triphosphates.!! ! 3.! Below!is!a!schematic!of!gene!Y,!which!encodes!protein!Y.!The!promoter!region!is!indicated!by!the! dotted!line!at!position!L100!to!0.!Transcription!begins!immediately!following!the!promoter,!at!position!1.!! !

! ! a)!Approximately!how!many!nucleotides!long!will!the!primary/nascent!transcript!be?!!From!1L5000!plus!add! the!polyA!tail!(typically!200L250!Adenines).! b)!Two!different!mature!transcripts!are!produced!from!this!gene,!one!is!approximately!2000!nucleotides! long,!the!other!is!approximately!3000!nucleotides!long.!Explain!how!two!different!transcripts!can!be! produced!from!this!single!gene.!Alternative!splicing! c)!Is!this!gene!likely!to!be!expressed!in!a!eukaryotic!cell!or!prokaryotic!cell?!Explain!your!thinking.! Eukaryotic!cell,!bacteria!don’t!have!introns!and!no!splicing!machinery!(splicesome).! ! d)!Assume!gene!Y!was!mutated!such!that!the!base!pair!found!at!position!100!was!changed!from!an!A!to!a! G.!Would!the!new!transcript!be!the!same!length,!shorter!or!longer!than!the!transcript!produced!from!the!wild! type!gene!Y?!Give!all!possible!answers!and!explain!your!thinking.!Transcript!will!be!the!same!length,!as! RNA!polymerase!does!not!care!about!the!sequence!it!transcribes.!! ! e)!Would!the!protein!produced!by!the!mutant!gene!Y!be!the!same!length,!shorter!or!longer!than!the!protein! produced!from!the!wild!type!gene!Y?!Give!all!possible!answers!and!explain!your!thinking.!Most!likely! shorter,!because!the!first!AUG!start!codon!cannot!be!used!(mutated).!The!ribosome!will!continue!scanning! until!another!AUG!is!found.!! If!another!AUG!is!found,!the!ribosome!may!be!reading!the!codons!in!a!different!reading!frame.!! The!protein!may!be!longer!if!translation!starts!in!a!different!reading!frame!and!in!this!new!reading!frame!a! stop!codon!is!further!away!from!the!start!codon.! ! f)!Briefly!describe!at!least!3!different!types!of!regulatory!DNA!and!name!the!proteins!that!interact!with!each.! DNA!replication:!origin!in!DNA!and!initiator!proteins! Transcription:!Promoter!with!TATA!box!(bound!by!transcription!factors)!and!enhancers!bound!by!activator! proteins.!!

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4.!Shown!below!is!the!schematic!of!Gene!B.!The!numbers!within!the!boxes!indicate!the!length!(in!base! pairs)!of!each!region.!The!DNA!sequence!corresponding!to!the!translational!start!and!the!stop!codons!are! indicated!

! You!observe!that!Gene!B!is!transcribed!both!in!epidermal!and!muscle!cells!to!produce!a!nascent!/!primary! mRNA!transcript.!This!mRNA!directs!the!synthesis!of!two!different!proteins!in!these!two!different!cell!types.!! In#the#muscle#cells#Gene#B#encodes#a#protein((100#amino#acids#long)#that#functions#as#a#nuclear( protein((NP01)(! •! In#epidermal#cells,#Gene#B#encodes#a#protein((200#amino#acids#long)#that#functions#as#a#single#pass# transmembrane#protein#.#! •!

Could!Gene!B!direct!the!synthesis!of!two(different(proteins(due!to...!!

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Splicing:!explain!why!or!why!not!yes,!alternative!splicing!could!result!in!either!keeping!an!exon!with!20L30! hydrophobic!amino!acids,!giving!the!protein!the!ability!to!pass!through!the!membrane!as!an!alpha!helix! Promoter!sequence:!explain!why!or!why!not!no,!that!would!only!determine!whether!the!RNA!transcription!is! initiated!or!not.!! ! 4.!You!want!to!study!another(nuclear(protein,(NP=2(in!mouse!muscle!cells.!You!identify!a!mutant!cell!line,! which!shows!a!cytosolic(location(of(NP=2(in!muscle!cells.!The!amino!acid!lysine,!at!position!60!(lys!60 ! encoded!by!5’AAG3’(codon)!of!NPL2!is!critical!for!its!function.!The!following!is!a!schematic!of!the!tRNA!

i.!Label!the!5'!and!3'!ends!of!the!tRNA!by!filling!in!the! shaded!boxes.!5’!Phosphate!group,!3’!OH! ii.!In!the!blank(box,(write!the!anti=codon(that!base!pairs! with!the!5’AAG3’!codon!for!Lys.(5’=CUU=3’! iii.!Is!the!tRNA!in!the!schematic!charged#or!uncharged?! Explain(why!you!selected!this!option.!Uncharged,!no!AA! covalently!bound!(via!ester!bond)!to!3’OH!of!tRNA! iv.!If!you!chose!uncharged,!please!draw!where!the!amino! acid!would!be!covalently!bound!to!the!tRNA.!To!3’OH,! amino!acid’s!carboxyl!group!is!attached!to!the!RNA,! leaving!the!amino!group!free.!

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specific!for!lys60 .!

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5.!Here!is!a!model!of!the!three!stages!of!translation.!This!model!contains!at(least(four(mistakes,! please!help!identify!those!mistakes.!In!addition,!please!label!the!NLterminus!and!CLterminus!of!the! polypeptide!and!the!3’!and!5’!end!of!one!tRNA.!

6.!Protein!synthesis!is!a!key!target!for!antibioticLmediated!treatment!of!bacterial!infections.!In!fact,! antibiotics!have!been!identified!that!inhibit!almost!every!step!of!the!translation!cycle.!Why!do!antibiotics!not! harm!our!own!cells?!There!are!slight!differences!in!the!proteins!and!rRNAs!that!make!up!the!ribosome!and! differences!in!initiation!factors!that!we!can!target!with!various!drugs. For!each!of!the!antibiotics!described!below,!explain!what!you!would!expect!to!see.! Tetracycline!and!streptomycin!interfere!with!binding!of!charged!tRNAs!to!the!A!site!of!the!ribosome.!!

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No!protein!would!be!synthesized.!Initiation!would!start,!with!the!fLMet!tRNA!in!the!P!site,!and!the!process! would!stall!out!from!here.!! Chloramphenicol!interferes!with!peptide!bond!formation.!Charged!tRNA!would!sit!in!A!site,!but!if!no!peptide! bond!can!be!formed,!elongation!stalls!out!here.!!! Erythromycin!interferes!with!translocation!of!the!tRNAs.!After!peptide!bond!is!formed!between!fLMet!in!P! site!and!new!charged!tRNA!in!A!site,!the!ribosome!would!sit!there!and!cannot!be!translocated!down!3! nucleotides!along!the!mRNA,!elongation!stalls!out!here.!!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Transcription+ Translation+ Replication+ Product! RNA! Protein/polypeptide! DNA! ! Monomeric! NTPs! Amino!acids,!charged!tRNA! dNTPs!!!Deoxy!ribonucleotide! building!block! Ribonucleotide!triphosphates! triphosphates! Polarity!of!the! NH2!terminal!to!COOH! 5’à฀!3’! 5’à฀!3’! product! terminal! ! Chemical!bond! Phosphodiester! Peptide!bond! Phosphodiester! between! monomers! Site!of!initiation! Promoter!(L10!and!L35!box!in! AUG!on!mRNA! origin! ! bacteria)!! ! Promoter!with!~L25!TATA!box! and!enhancers!in!eukes! Initiation! E.!coli:!sigma!factor!plus!RNA! Initiation!factors!(IFs),!small! Initiator!proteins!bind!origin! proteins! polymerase! subunit!of!ribosome!(with! Then!helicase!unwinds,!SSBPs! ! ribosomal!binding!site!in!16S! hold!DNA!open,!primase! Eukaryotes:!transcription! ! rRNA),!fLMetLtRNA! makes!RNA!primer! factors,!activator!proteins! ! and!mediator!complex!plus! ! RNA!polymerase! Elongation! RNA!polymerase! Ribosomal!subunits,! DNA!polymerase!III,!sliding! proteins! elongation!factors!(EFs,!Tu! clamp,!helicase,! ! and!G),!charged!tRNAs.! topoisomerase!I,!SSBPs,! ! Ribozyme!=!23S!rRNA! primase!for!lagging!strand,! ! catalyses!peptide!bond! DNA!pol!I!and!ligase!! ! formation!

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Termination! ! ! ! ! ! Location!in!cell! ! ! ! ! Types!of! processing,! packaging!!or! modification! product!can! undergo!

E.!coli:!mRNA!has!hairpin! followed!by!string!of!Us! ! Eukaryotes:!!AAUAAA,!then! cleave!and!add!poly!A! E.#coli!:!cytoplasm! ! Eukaryotes:!!nucleus!

Eukaryotes!=!5’cap,!polyA! tail,!splicingLintron!removal!

Stop!codon!(UAA,!UAG,!UGA)! DNA!ligase!and!topoisomerase! and!release!factor!protein! II!in!bacteria!Ultimately! termination!site!on! chromosome!(reaches!end!of! circular!DNA!or!ends!of!linear! DNA)! E.#coli!!Cytoplasm! E.#coli!:!cytoplasm! Eukaryotes:!Cytoplasm!(or!in! ! endoplasmic!reticulum)! Eukaryotes:!nucleus!

Degradation,! phosphorylation,! glycosylation,!disulfide! bonding,!cleavage!

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Eukaryotes!=!chromatin!–!DNA! packaged!w/!histones!and! other!proteins!to!make! nucleosomes,!30nm!fiber!and! condensed!mitotic! chromosomes!...