Midterm #2: Transcription Flashcards Preview

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Flashcards in Midterm #2: Transcription Deck (46):

What is transcription?

  • The synthesis of RNA
  • genetic information that is stored in DNA to a template that can be read by the ribosome to synthesize protein


DNA is trancribed by enzymes called...

  • RNA polymerases (RNAP)
  • step-by-step addition of ribonucleoside monophosphates to a RNA chain
  • RNAP are template directed enzymes 


E. coli RNAP Requirements

  • Ribonucleoside triphosphates 
  • Magnesisum ion
  • single-stranded DNA template
  • DOES NOT require a primer strand
    • Can initiate transcription de novo

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E. coli RNAP complex

  • 450 kD
  • RNA Core Enzyme: a2BB'w (no sigma subunit)
  • RNA Holoenzyme:  a2BB'w plus sigma 

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Where does transcription initiate?

  • Transcription of DNA template begins at a specific promoter
  • Promoter has -35 sequence (TTGACA) and -10 sequence (also known as Prinbow Box) (TATAAT)

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What is a gene?

  • segment of DNA that is transcribed for the purpose of expressing the encoded genetic information as a protein 


How do promoters control the rate of transcription?

  • ~2000 promoters per E. coli chromosome. The consensus sequence has been derived by examining the sequences of 100's of promoters 
  • "Strong Promoters": sequences match closely to the consensus sequence and can initiate RNA synthesis as often as every 2 seconds
  • "Weak Promoters": ​differ in one or more of the consensus nucleotides; the more differences the weaker the promoter. Weak promoters may only initiatiate RNA synthesis once every 10 minutes

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Steps of Transcription Initiation 

  • RNAP holoenzyme binds to DNA and scans the duplex for a promoter 
    • RNAP core enzyme is highly processive but it cannot initiate RNA synthesis 
  • Sigma subunit specifically recognizes the promoter sequence and stops to form the closed promoter complex
  • RNAP unwinds ~17 bp of DNA to form open promoter complex
  • After 8-10 nucleotides are added, the sigma subunit dissociates from the holoenzyme. This is called promoter clearance and signals the transition from the initiation phase to the elongation phase of transcription.  

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Enlongation and Termination of Transcription

  • RNAP core enzyme is highly processive 
  • transcription bubble: area that contains RNAP core enzyme, DNA and the nascent RNA transcript 
  • Synthesized at rate of 40 bp/sec
  • RNA polymerase lacks nuclease activity (i.e. no editing function) and therefore the fidelity for RNA syntheis is 10-4 to 10-5 error/bp. 
  • The end of the gene contains stop signals
    • termination can be p-dependent or p-independent
  • Teremation stop signals lie in the newly synthesized RNA strand rather than the DNA template 

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Transcription Initiation is Highly Regulated in Prokaryotes

  1. Promoter sequences and sigma factors
    • ​A variety of different promoter sequences, each with a preferred sigma factor
  2. Repressors and Inducers
    • ​Proteins and small molecules that can prevent or enhance transcription of a gene or genes 
  3. ​Catabolite Repression 
    • ​A special case - repress many metabolic genes in glucose-rich environments 


Sigma Factors Enable Regulation of Transcription 

  • Prokaryotics cells contain only one core RNAP, but multiple sigma factors
  • Each sigma factor regonizes a specific set of promoter sequences - this allows bacteria to regulate patterns of gene expression by using different sigma factors


Different Sigma Factors and their Biological Roles

  • Sigma 70
    • "housekeeping" sigma factor, transcribes most genes in growing cells
  • ​​Sigma 38
    • the starvation/stationary phase sigma factor
  • Sigma 32
    • the heat shock sigma factor
  • Sigma 24
    • the extracytoplasmic stress sigma factor 

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Heat Shock Response

  • An abrupt increase in the environmental temperature results in the synthesis of heat shock proteins in E. coli 
  • The promoter regions for the heat shock genes have divergent -10 and -35 sequences. A different sigma factor (sigma 32) recognizes this promoter. 
    • Sigma 32 only activated at elevated temperature 

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Bottom Line of Transcription and Sigma Factors

  • RNAP core enzyme can associate with a variety of different sigma factors to yield a variety of holoenzymes that recognize specific promoters in the E. coli genome  

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The lac operon: Repressor

  • ​Transcription of the lac genes affords three proteins required for lactose utilization in E. coli
  • The lac repressor is a protein expressed from the LacI gene; it binds to the operator site (O) and prevents RNAP from binding to the promoter site (P)
  • Thus the lac repressor is a negative regulator of transcription 

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The lac operon: Inducer

  • ​Lactose is metabolized to allalactose by E. coli, which binds to the lac repressor
  • the repressor-allactose complex dissociates from DNA, which allows RNAP to bind to the promoter region of the lac operon, and stimulates transription 
  • Allalactose is an inducer

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Catabolite Repression 

  • Abundance of glucose decreases the expression of genes specifying proteins involved in the metabolism of other catabolites (lactose, arabinose, and galactose)
  • Cyclic AMP serves as a "hunger signal" and stimulates the initiation of transcription of the lac operon 
  • cAMP produced by adenylate cyclase, which is inhibited by glucose (negative allosteric regulation)
  • As long as glucose levels are plentiful, adenylate cyclase is inhibited and cAMP levels are low
  • But, when glucose levels drop, adenylate cyclase is no longer inhibited and cAMP syntheis proceeds

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Name this structure

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Name this structure

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Catabolite Repression: CAP

  • cAMP binds to protein known as Catabolite Gene Activator Protein (CAP) and enhances it's affinity for DNA (positive allosteric regulation)
  • The CAP:cAMP complex binds to the promoter region of the lac operon and stimulates transcription 
    • lacP deviates significantly from the constitutive promoter sequence, and is therefore a "weak" promoter (i.e. baseline transcription is slow)
  • The CAP:cAMP complex is a positive regulator (it turns transcription on)
  • Note that the presence of the the lac repressor inhibits transcription from the lac operon no matter what 


Rifamycin B

  • Produced by Streptomyces mediterranei 
  • Semi-synthetic derivative rifampicin 
  • Inhibits prokaryotic transcription
  • Inhibits first phosphodiester bond by the RNA polymerase 
  • Treats Mycobacterium tuberculosis infections


Name this structure 

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Eukaryotic RNA Polymerases

  • Large (>500kDa), multiprotein complexes
  • Same catalytic requirements as for the prokaryotic RNAP
  • Type I:
    • Location: Nucleolus
    • Cellular Transcripts: 18s rRNA, 5.8s rRNA, 28s rRNA
  • Type II:
    • Location: Nucleoplasm
    • Cellular Transcripts: mRNA, hnRNA, snRNA
  • Type III:
    • Location: Nucleoplasm
    • Cellular Transcripts: tRNA, and 5s rRNA

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Eukaryotic Promoters and Transcription Factors compared to Prokaryotics

  • like prok, euk RNAP cannot initiate transcription by themselves
  • NO sigma factors 
  • Transcription factors bind DNA and recruit RNAP II to promoter 
  • Euk promoters are more complex

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  • analgolous to Prinbow box (-10 sequence), but located further upstream


Eukaroytic Promoters and Transcription Factors

  • TF bind to the promoter and stimulate mRNA transcription by RNA polymerase
  • The general transcription factors (over 20 proteins) assemble at the TATA box to form a pre-initiation complex
  • Basal transcription complex is necessary, but not sufficient for most eukaryotic promoters
  • Once assembled, the RNAP initates RNA synthesis and departs from the prmoter, leaving the TF behind. 

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Name this structure

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Amanita phalloides

  • Poisonus mushroom that causes 95% of mushroom fatalities
  • Diarhea and cramps...remission...4-5 days later is failure in liver and kidneys
  • 15% die in 10 days
    • comotose, renal failure, liver failure, hepatic coma, respiratory failure, death
  • Toxin alpha-amanitin, potent inhibitor of eukaryotics RNAP II, inhibits elongation 


Posttranslational mRNA Processing: 5' Capping


  • Posttranslation mRNA processing is only in euk
  • Modified G attached to 5' end  of transcript through rare 5'-5' triphosphate bond 
  • Slows down degradation, enable nuclear export, and promotes translation 


3' Polyadenylation

  • A string of A's is added to the 3' end of the transcript 
  • Also slows down degradation, and assists in export and translation



  • Intron are removed from mRNA and the exons are joined together
    • noncoding vs. coding
  • Alternative splicing can generate mutliple distinct mature mRNA from a single gene. 
  • Carried out in large ribonucleoprotein complex called the splicesome 


Transcription is even more regulated in EUK compared to PRO​ (6)

  1. Promoters, transcription factors and effectors
  2. Enhancer:activator and Silencer:repressor interactions
  3. Chromatin remodeling
    • ​Chromosomal DNA is tightly packed into arrganement called chromatin, and must be unpacked for efficent transcription
  4. ​DNA methylation
    • ​Cytosine methylationcan downregulate the transcription of a stretch of DNA
  5. ​mRNA processing (posttranscriptional)
  6. microRNA regulation (posttranscriptional)
    • Expression of small, non-coding RNA transcripts can downregulate the translation of specific mRNAa 


Transcription Elements

  • In the -40 to -150 upstream region 
  • numerous compared to prok -35 element
  • Each element recognized by specific TF
  • Activation of specific gene require not only that transcription factor elements be present in the DNA, but also that the required protein be expressed in the cell. 


Transcription Elements Examples

  • Element: TF
  • GC Box: SP1
  • CAAT Box: CTF
  • Octamer motiffs: Oct-1 and Oct-2

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Binding of TF to DNA can be allosterically regulated by..

  • an effector molecule and/or by phosphorylation

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Enhancer, Activator, Silencers and Repressors

  • Many euk genes also posses transcriptional control elements called enhancer. They can be several thousand bp upstream or downstream and their orientation does not matter
  • Activators are TF that bind enhancer and aid in RNAP recruitment to the promoter
    • cf. the e. coli CAP protein
  • silencer is a DNA element that binds a repressor protein and downregulates  transcription 
    • cf. the lacO sequence and the lac repressor protein 


Regulation of Transcription Initiation: In a Nutshell

  • Complicated 
  • Assembly of pre-initation complex and transcription initiation involves dozens of proteins- each of which incrementally stabailze the complex and thus promote transcription by RNAP II 

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Chromatin, Nucleosome, Histone

  • DNA plus protein=chromatin
  • Fundamental unit of chromatin is nucleosome
  • Core particle is 146bp DNA wrapped around octomer of histone proteins
    • ​(H2A-H2B)2(H3)2(H4)2
  • Histones have lots of Lys and Arg that interact with DNA negative phosphte backbone
  • Little sequence specificity in nucleosome formation
  • 75% of the DNA surface is asscebile to DNA binding proteins 

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How is chromatin compacted?


  • A linker DNA (8-114 bp) connects nucleosomes so they look like bead on a string
    • polysomes
  • Histone H1 associates with linker DNA to bring neighbooring nucleosomes close together to form the 10 nm nucleofilament 
  • The 10 nm nucleofilament further compacts into a 30 nm fiber, which can be further compacted into the chromosome structure 

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Nuclesomes can block acess to the promoter. In chromatin remodeling, the histones are modified so as to regulate gene expression by:

  • creating binding sites for regulatory proteins 
  • by freeing the DNA from tightly packed nucleosomes so that it is more accessible to binding proteins 


Types of histone modification and which residues:

  • ​Acetylated
    • Lys
  • Methylated
    • Arg/Lys
  • Phosphorylated
    • Ser/Thr
  • ADP-ribosylated
    • Arg/Asp/Glu
  • Ubiquinylated
    • Lys
  • Sumoylated
    • Lys



  • Histone acetyltransferases (HAT)
    • add acetyl groups to Lys
    • neturelizes charge
    • trasncriptional activation
    • ​Often part of multisubunit TF
  • Histone deactylases (HDAC)
    • remove acetyl groups
    • gene silencing 

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Name this Structure

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DNA methylation

  • 5-methylcytosine (m5C) only methylated base in human DNA
  • mostly in Cytosine-phosphate-Guanosine (CpG) sequences
  • around promoter regions, known as CpG Islands
  • Methylation in CpG islands interefers with TF binding
    • switches genes off
  • methylcytosine binding protein can bind and repress the gene
  • methylation patterns passed down to progeny, known as epigenetic inheritance


MicroRNA (miRNA) Regulation of Gene Expression

  • miRNA: small, noncoding RNA that regulates gene expression through RNA interference (RNAi)
  • Primary (pri-)miRNA is cleaved by Drosha into ~70 bp pre-miRNA and exported into the nucleus 
  • Dicer then cleaves the pre-miRNA into ~22 bp small interfering (si)RNA 
  • RISC (RNA-induced silencing complex) binds siRNA, releases the "passenger strand" and mediates formation of a duplex between the "guide strand" and complementary (target) mRNA. 
  • This complex blocks translation and stimulates degradation of the polyA tail  

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Biological Importance of RNAi

  • human genome consists of >1000 miRNA's, which regulate 60% of all genes
  • Similar process in the recognition of RNA viruses
  • DS RNA is recognized by dicer and cleaved into siRNA
  • RNAi could be used clinically to selectively knock down particular genes (cancer, anti-viral therapy, degenerative diseases)

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