Midterm #2: Transcription Flashcards
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
- Can initiate transcription de novo

E. coli RNAP complex
- 450 kD
- RNA Core Enzyme: a2BB’w (no sigma subunit)
- RNA Holoenzyme: a2BB’w plus sigma

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)

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

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.

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

Transcription Initiation is Highly Regulated in Prokaryotes
- Promoter sequences and sigma factors
- A variety of different promoter sequences, each with a preferred sigma factor
- Repressors and Inducers
- Proteins and small molecules that can prevent or enhance transcription of a gene or genes
- 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
- the extracytoplasmic stress sigma factor

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

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

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

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

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

Name this structure

ATP
Name this structure

cAMP
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

rifampicin

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

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











