Midterm #2: Transcription

Question 1

What is transcription?

Answer 1

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

Question 2

DNA is trancribed by enzymes called…

Answer 2

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

Question 3

E. coli RNAP Requirements

Answer 3

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

Question 4

E. coli RNAP complex

Answer 4

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

Question 5

Where does transcription initiate?

Answer 5

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

Question 6

What is a gene?

Answer 6

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

Question 7

How do promoters control the rate of transcription?

Answer 7

• ~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

Question 8

Steps of Transcription Initiation

Answer 8

• 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.

Question 9

Enlongation and Termination of Transcription

Answer 9

• 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

Question 10

Transcription Initiation is Highly Regulated in Prokaryotes

Answer 10

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

Question 11

Sigma Factors Enable Regulation of Transcription

Answer 11

• 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

Question 12

Different Sigma Factors and their Biological Roles

Answer 12

• 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
    ​

Question 13

Heat Shock Response

Answer 13

• 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

Question 14

Bottom Line of Transcription and Sigma Factors

Answer 14

• 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

Question 15

The lac operon: Repressor

Answer 15

• ​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

Question 16

The lac operon: Inducer

Answer 16

• ​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

Question 17

Catabolite Repression

Answer 17

• 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

Question 18

Name this structure

Answer 18

ATP

Question 19

Name this structure

Answer 19

cAMP

Question 20

Catabolite Repression: CAP

Answer 20

• 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

Question 21

Rifamycin B

Answer 21

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

Question 22

Name this structure

Answer 22

rifampicin

Question 23

Eukaryotic RNA Polymerases

Answer 23

• 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

Question 24

Eukaryotic Promoters and Transcription Factors compared to Prokaryotics

Answer 24

• 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

Question 25

TATA Box

Answer 25

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

Question 26

Eukaroytic Promoters and Transcription Factors

Answer 26

• 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.

Question 27

Name this structure

Answer 27

alpha-amanitin

Question 28

Amanita phalloides

Answer 28

• 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

Question 29

Posttranslational mRNA Processing: 5’ Capping

Answer 29

• 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

Question 30

3’ Polyadenylation

Answer 30

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

Question 31

Splicing

Answer 31

• 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 **

Question 32

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

Answer 32

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
     ​​

Question 33

Transcription Elements

Answer 33

• 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.

Question 34

Transcription Elements Examples

Answer 34

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

Question 35

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

Answer 35

• an effector molecule and/or by phosphorylation
  *

Question 36

Enhancer, Activator, Silencers and Repressors

Answer 36

• 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
• A silencer is a DNA element that binds a **repressor **protein and downregulates transcription
  
  • cf. the lacO sequence and the lac repressor protein
    ​

Question 37

Regulation of Transcription Initiation: In a Nutshell

Answer 37

• 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

Question 38

Chromatin, Nucleosome, Histone

Answer 38

• 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

Question 39

How is chromatin compacted?

Answer 39

• 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

Question 40

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

Answer 40

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

Question 41

Types of histone modification and which residues:

Answer 41

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

Question 42

HAT and HDAC

Answer 42

• 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

Question 43

Name this Structure

Answer 43

5-Methylcytosine

Question 44

DNA methylation

Answer 44

• 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

Question 45

MicroRNA (miRNA) Regulation of Gene Expression

Answer 45

• miRNA: small, noncoding RNA that regulates gene expression through RNA interference (RNAi)
• Primary (*pri-*)miRNA is cleaved byDrosha** 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

Question 46

Biological Importance of RNAi

Answer 46

• 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)