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Transcription occurs in the ______in eukaryotes and translation in the _____.
Nucleous; cytoplasm
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In prokaryotes transcription and translation occur.....
Simultaneously, once the mRNA is made translation begins
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Where does transcription take place in prokaryotic cells?
Cytoplasm
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RNA polymerase synthesizes a _____ copy of _____
RNA;DNA
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What forms the protein complex at the start site of transcription?
RNA pol and transcription factors
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Genomic DNA is....
Double stranded and encodes multiple genes
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Contains all info for correct regulation, specifies start site of transcription
Promoter
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What does precursor RNA make
- mRNA and translated it to proteins
- other RNAs (rRNA, snRNA)
- involved in RNA processing, transclation, mRNA stability and transcriptional control
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Proteins and mRNA can _____ transcription and ______ to the final product
regulate; maturation
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Genes are orginized into
operons
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mRNA is polycistromic and gives rise to...
multiple proteins
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Controlled by proteins that activate or repress transcription
Promoter
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E. coli RNA pol....
- acts like a zipper and unwinds DNA (17bp)
- non-template strand displaced outside
- RNA synthisized inside
- covers about 35 bps
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____ removes negative supercoils and _____ removes positive supercoils
Topo I; Topo II
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RNA pol subunits
- 2 alphas
- beta
- beta'
- omega
- sigma factor
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The BB' contains the _______ and has _______ activity
Acitve site; enzymatic
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RNA pol
- Can bind and catalyze RNA but has no specificity
- Can't recognize promoter
- Recognition by the sigma factor
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Regulates binding to the promoter, has multiple units, and may act to stabilize the enzyme
Sigma factor
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Interacts with other TF
Alpha
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Important Sigma factors
- 70: normal; most abundant
- 32: heat shock
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E. coli propoter contains:
- region @ -35 and +10 bound by sigma factor
- the UP element
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Consensus DNA binding sites
the most common nucleotide in each position in hypothetical binding sequences
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Heat shock promoters
- signa 32 recognized promoters
- UP is pound by RNA subunit
- binding sites have different sequences
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How does initation start?
- Pol promoter binds to DNA in the major groove and slides down the DNA or jump from position to position
- sigma factors binds tightly
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Initiation of transcription
synthesized from the 5' end using the template strand
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Promoter Clearence
- sigma factor no longer interaxts with DNA specifically after synthesis of first 8-9bp
- ofter released, sometimes noves with pol
- bubble increased to 17bp
- RNA/DNA hybrid forms; ~8bp
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Rho independent termination
- string of U's at 3' end weakens duplex
- hairpin formation, 15-20 bases before end of strand causes pol to fall off
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Rho dependent termination
- no A's in template
- sometimes forms a haripin
- Rho binds to the loading site on RNA and migrates to termination site
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How doeslactose get into E. coli cells
transported by lac permerase
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Lactose is converted into ______ and ______
glucose; galactose (sometimes allolactose first)
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Lac operon
- Produces mRNA encoding 3 orf's
- mRNA controlled by activity of promoter
- repressor produced from own mRNA and promoter P
- Repressor binds to O1,O2, and O3 ihibiting lac operon expression
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Lac repressor
- made continuously
- binds pair of operator sequences and inhibits transcription
- basel livel of beta gal and lac permease at all times
- essential for lac operon control
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In the presence of lactose
- Allolactose is produced and binds to the lac repressor
- prevent lac repressor from binding to DNA
- lac promoter expression increades
- basel expression of lac permease and beta-gal is essential to the control
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Negative regulation
- Repressor bound to the DNA no transcription
- Modulator bound to repressor away from DNA = transcription
- Modulator bound to repressor on DNA = no transcription
- unbound repressor and modulator = transcription
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Repressor bound to DNA
- Binds as a dimer to operator seq
- 2 pairs associate to form a tetramer
- DNA forms loops around tetramer with tighest affinity for O3 represor
- The alpha helix binds in the major groove
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Allolactose's conformational change
withdraws the DNA binding domains preventing DNA binding
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Lac promoter in molecular biology
- Can be put in front of a gene of interest
- Little/no expression of this gene in the absence of an inducer
- Transcription is induced by addidion of artificial inducer IPTG
- Use to drive expression
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In a mormal E. coli cell with normal operon....
- in the presence of allolactoes beta gal with break it down
- inducer is removed
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Weak promoters
- in the presence of allolactose there is little expression of lac promoter
- -10 and -35 regions are diverged from the consenses seq and bind the sigma factor weakly
- no UP seq and low beta gal production
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In the well feed state, there is little _____ expression even in the presence of _______
lac operon; lactose
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During starvation
- lac operon is turned on and lactose is present
- allows use of lactose as a carbon source when othere perferential sources (glucose) are absent
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Starvation activates ______
CRP (cAMP receptor protein)
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CRP
- binds lac promoter and activates transcription if lactose is present
- binding is induced by cAMP
- CRP and cAMP is an example of postive regulation
- When bound => transcription
- binds as dimer and change DNA to palimdrome seq
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Low glucose, high cAMP
- cAMP binds to CRP
- lac repressor released
- RNA pol activated
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High glucose, low cAMP
- No CRP bound
- no transcription
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Positive regulation 1
- Activation: CRP bound alone = transcription
- Inactivation: CRP unbound but cAMP bound = no transcription
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Positive regulation 2
- Activation: CRP bound with cAMP = transcription
- Inactivation: CRP unbound alone = no transcription
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Polymerase I
- 45S per-ribosomal RNA synthesis
- occurs in the nucleous
- nucleolar processing to 18, 5.8, and 28S rRNAs
- requires snoRNAs
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Polymerase II
- mRNA synthesis, some snoRNAs
- occure in the nucleoplasm
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Polymerase III
- synthesis of tRNA, snRNA, and 5S RNA
- ocurrs in the nucleoplasm
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snRNA
small nucleolar RNA
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Pol II
- 10-14 subunits that perform the function of the 6 in bacterial RNA pol
- interact with other proteins involved in eukaryotic transcription
- cannot initiate transcription alone
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Large Pol II subunit
- 240 kDa (homoenzyme >500kDa)
- homologous to B' (second largest is same as B)
- CTD ontains 7 AA repeat: tyr-ser-pro-thr-ser-pro-ser (repeats ~50x)
- ser and thr can be phosphorylated
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Eukaryotic promoters
- specifies the start site
- specifies when (during development) and where (on the gene)
- regulate transcription of all RNA
- found 5' of transcription unit
- start region is internal
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Cis-acting promoters
encode on the same DNA
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Trans-action promoters
action by a factor; anything that acts on the gene to change transcription
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Proximal promoter
- region clsoe to the start site of transcriotion
- specifie the exact start site
- binding site of pol and basal TFs
- contains everthing that allows transcription
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Distal Promoter
- distand (away) form the start site
- regulates the rate of gene expression
- may contain enhancers
- responsible for the postion of pol II
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Enhancers
- element that works up/down stream in an orientation independent manner to regulate transcription
- the more proteins bound the better
- changes the structure to affect transcription
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TATA box
- TATAAA consensus (A-T rich region)
- not alway present
- mostly found in highly expressed genes
- binds to TFIID
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initiator
- found near TATA box
- unwinding site
- interacts with complexes containing TFIID
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downstream promoter & core elements
- not always present
- interacts with complexes containing TFIID
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Regulatory sequences
- binds lots of regulatory TF at once
- often 5' of TATA box but can be anywhere
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Pol II has no specific ______ for transcription
Start site
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Pol II activity is controlled by _____and _____ that bind to distant DNA elements.
basal transcription factors; other TFs
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Prokaryotic Promoters
- recognizes specific DNA seq
- interacts w/ proteins that bind locally
- contained w/in ~80bp
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TATA binding proteins (TBP)....
- binds the TATA box and specifies the start site for transcription
- occurs abot 25 bp downstream of TATA box
- part of TFIID
- binds in the minor groove and bends ~80 degrees
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TBP binding to DNA
- binds as a monomer w/ sudo-dimer character
- covalently bound
- 2-fold acix of symmetry
- binds to minor groove - induced fit
- no inherent directionality
- beta sheets bind and stabilized by alpha helix
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Transciption initiation occurs
via a closed complex where the factors bind as a chain of events
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Open complex
once all the proteins are bound, DNA start to unwind in the pol unit
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Promoter Clearance
- helicase activity of TFIIH
- phosporylation of CTD of polymerase
- TFIID and other proteins left behind
- TBP taken along with pol
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The 5' G-cap is found in ______
eukaryotes and varies by species
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5' cap
- protect against 5' degradation
- added at the start of transcription
- added to 5' mRNA via 5'-5' triphospate bond
- GTP is nethylated
- requires methyltransferase to add adoMet
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The primary transcript is the mRNA with ____ and ____ or is not ______
introns, exons, mature
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Primary transcript is considered the _______ product and contains about ________ bp
first finished, 100,000
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Mature mRNA has _____ removed
introns
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Splicing is the removal of _____ and joning of _____
introns, exons
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Splicing in mRNA
- requires small nuclear ribonucleoproteins (snRNPs)
- contain small nuclear RNAa (snRNA)
- 5 highly conserved snRNAs
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spliceosome assembly
- uses U1 and U2 to bring the exons together
- requires ATP to build up RNA complex that removes introns
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The Poly A tail _____eukaryotes and ______ prokaryotes
stablize, destablize
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Poly A tail
- added to the end, not encoded
- RNA transcribed past the pol A site
- AAUAAA pol A addition site located 10-30 baseds 5' of mature 3'end
- GU rich seq 20-30 bases downstream f 3' end
- large enzyme complex cleaves RNA and adds pol A 80-250 As)
- doesnt require template
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