Molecular Bio Midterm 3 (May 23)

• Transcription

  The process whereby genetic information in the DNA is used to create an RNA strand of complementary sequence
• Genome


  The sum of all genetic information contained in the DNA of an organism
• Transcriptome


  The sum of all the RNA transcripts in a cell in a given moment
• Every cell in your body has the same genome, but not every cell has the same transcriptome
• RNA


  Ribonucleic Acid
• Differences between DNA and RNA

  • RNA uses Uracil as one of its bases, DNA uses Thymine
  • RNA has a hydroxyl group on it's 2' carbon, DNA does not
• DNA


  • Can form a double helix, which is very regular and very stable
• RNA transcripts


  • Can serve as carriers of information from DNA to protein (i.e. messenger RNA, or mRNA)
  • Can form complex secondary and tertiary structures, giving it functional capabilities
• Types of RNA transcripts

  • mRNA - messenger RNA
  • rRNA - ribosomal RNA
  • tRNA - transfer RNA
  • Other non-coding RNAs (small nuclear RNAs, long non-coding RNAs, micro RNAs)
• Transcription in a Nutshell

  1. RNA polymerase binds to double stranded DNA

  2. DNA opens up to expose single strands

  3. RNA polymerase reads template strand and adds ribonucleotides (NTPs) to growing RNA chain

  4. DNA reforms double strand and exits, RNA chain exits
• Coding strand


  The reverse complement of the Template DNA strand
• The RNA transcript sequence is identical to the coding strand (except T is U)
• 5'


  The beginning of the transcript
• 3'


  The end that the polymerase is progressing towards
• The last base to be transcribed is the 3' end
• RNA polymerase


  • Bacteria have 1 RNA polymerase
  • Eukaryotes have 3 (RNA Pol II is the one most similar to bacteria)
• Transcription Bubble


  • DNA duplex is unwound into single strands for a moving "bubble" of about 17 base pairs
  • Inside this bubble, the polymerase reaction occurs and the growing RNA chain is paired with the template strand to form a DNA/RNA hybrid (about 8 bp long)
  • As RNA polymerase moves through the DNA, it rotates the DNA, creating supercoils both in front and behind it
• Sigma Factor


  Bacterial protein that links RNA polymerase to the transcriptional start site at the front of a gene
• Common Sigma Factors


  • V70 - Housekeeping genes, most genes
  • V38 - Starvation genes
  • V32 - Heat shock
  • V54 - Nitrogen uptake and metabolism
• Promoter


  Region of DNA at the start of a gene, whose sequence determines where transcription begins and when
• Promoter regions


  • Consensus sequence - region in the promoter where sigma factor binds
  • -35 and -10 regions both contain consensus sequences
  • Upstream promoter (UP) element - AT-rich sequence, recognized by a subunit of RNA polymerase, increases binding and transcription
• Footprinting Assay


  1. Bind proteins (i.e. Sigma Factors) to DNA, then chop up the DNA, and see which regions are protected

  2. Regions of DNA bound by sigma factor/RNA pol will not be cut
• The sigma factor that binds to the osmY promoter is sigma 70
• Steps of Transcription in Bacteria

  • Initiation
  • Elongation
  • Termination
• Transcription Initiation, Part 1

  1. RNA polymerase holoenzyme (core + sigma factor) binds to DNA at promoter consensus sequence

  2. A structural change causes the DNA to melt (separate into two strands), and enter the polymerase active site, creating an transcription-competent open complex
• Abortive Initiation


  The structure is unstable for the first 8-10bp and will spontaneously stop and leave the channel
• Transcription Initiation, Part 3

  1. Sigma factor is initially blocking the exit channel for RNA

  2. The growing transcript will dislodge sigma factor from the exit channel, causing sigma factor to release from the polymerase

  3. Once released, the complex exits the promoter and initiation is complete
• Kinetic and Nucleic Proofreading

  • Two types of proofreading: 1) Detects mismatches immediately after phosphodiester bond formation and reverses the catalytic reaction, 2) Polymerase backtracks and nuclease activity hydrolyzes bond upstream of mismatched base
• Transcription Termination


  • Rho-Independent
  • Rho-Dependent
• Rho-Independent Termination


  Requires two sequences in the RNA: 1) Termination sequence in RNA about 15-20 nucleotides from end of RNA, creates hairpin structure, 2) AAA in template strand creates UUU in RNA strand, hairpin dislodges RNA at AAA/UUU interface
• Rho-Dependent Termination

  • rut site - rho utilization site, sequence on RNA that recruits rho
  • rho (U) helicase - helicase that travels along RNA transcript, when it encounters polymerase will hydrolyze ATP to dislodge transcript and shut down transcription
• During transcription, mRNA is produced by copying one strand of DNA as a template using complementary base pairing between nucleotides.
• The DNA polymerase III holoenzyme is the primary replicative enzyme that synthesizes new strands during DNA replication.