MICRB 316 slide 1

Page 1

• 200 nm

Page 2: Introduction to Genes

• Major Activities of Genes:

  • Information Repositories:

    • Genes store information: DNA → RNA → Proteins

  • Replication:

    • Genes can replicate unchanged over time

  • Mutations & Recombination:

    • Accepts occasional changes

• Gene Expression:

  1. Transcription:

     • Steps: Initiation, elongation, termination

     • Involves RNA Polymerase (RNAP)

  2. Translation:

     • Steps: Initiation, elongation, termination

     • Involves ribosomes

Page 3: RNA Polymerase (RNAP)

• Discovered in 1960 in animals, plants, & bacteria

• E. coli RNAP:

  • First studied bacterial RNAP

  • By 1969, subunits characterized on SDS-PAGE:

    • b: 150 kDa

    • b’: 160 kDa

    • a (2x): 40 kDa

    • s: 70 kDa

• Ion exchange chromatography separates s.

Page 4: Subunit Separation of E. coli RNA Polymerase

• Cartoon illustrating subunit separation by SDS-PAGE

  • Lane 1: holoenzyme

  • Lane 2: core enzyme after removal of s

  • Lane 3: o

Page 5: Structure of RNAP

• Key Components:

  • B’, a, core enzyme, and sigma factor (-35 and -10 regions)

Page 6: T4 Phage Transcription Phases

• Bautz's Findings:

  1. Holoenzyme transcribes specific classes of T4 genes

     • Immediate Early genes blocked by Imm. Early host RNAP (0-2 min)

     • Delayed Early & Late phases involve T4 protein synthesis (2-25 min)

  2. Core Enzyme Specificity:

  • Lacks specificity without s; only basic RNA synthesis

  • Adding s restores ability to transcribe unnicked DNA

Page 7: Forms of RNA Polymerase

• Types:A. Core EnzymeB. Holoenzyme

• Relative Transcription Activity:

  • T4 (native, intact): Core: 0.5, Holoenzyme: 33.0

  • Calf thymus (nicked): Core: 14.2, Holoenzyme: 32.8

Page 8: Nicked vs. Unnicked Transcription

• Transcription Characteristics:

  • Tn begins non-specifically at nicks without s; at specific sites with s

  • Promoter sites: Previously known as "Bautz sites"

• Study by Hinkle & Chamberlain (1972):

  • Nitrocellulose filter binding studies with core & holoenzyme

  • Assesses RNAP-DNA binding tightness

Page 9: Holoenzyme vs. Core Binding

• Results:

  • Holoenzyme binds DNA more tightly than core

  • Provides specific binding capability

Page 10: Reverse Binding Procedure

• Hinkle & Chamberlain Process:

  • Bind RNAP & unlabelled DNA; add excess labeled DNA

  • Determines RNAP-DNA association tightness based on radioactivity

Page 11: Binding Findings

• Radioactivity appeared simultaneously for both holoenzyme and core

Page 12: Transcription Initiation Dynamics

• Tight vs. Loose Binding:

  1. Tight sites initiate transcription immediately

  2. 8 tight binding sites (T7 early promoters) found

  3. Increased temperature improves holoenzyme tight binding

Page 13: Summary of RNAP Activities

• A. RNAP core + s + DNA interact

• B. RNAP holoenzyme initially binds loosely, scans for promoter

• C. Closed promoter complex still in dsDNA form

• D. DNA melting leads to open promoter complex, requiring s for tight binding.