Molecular Biology - Prokaryotic Transcription Notes

Prokaryotic Transcription

• Transcription is the process of synthesizing RNA from a DNA template, occurring in the 5' to 3' direction on a 3' to 5' DNA template.
• Coding strand: The DNA strand with the same sequence as the mRNA, related by the genetic code to the protein sequence.
• RNA polymerase: The enzyme synthesizing RNA using a DNA template (DNA-dependent RNA polymerase).
  • Function: copies one strand of duplex DNA into RNA.

Key Components

• Promoter: A DNA region where RNA polymerase binds to initiate transcription.
• Terminator: A DNA sequence causing RNA polymerase to terminate transcription.
• Transcription unit: The DNA sequence between initiation and termination sites for RNA polymerase.
  • May include more than one gene.
• Startpoint: The DNA position corresponding to the first base incorporated into RNA.
• Transcription unit: DNA sequence transcribed into a single RNA, starting at the promoter and ending at the terminator. No primer is needed, synthesis occurs in the 3' direction.

Directionality

• Upstream: Sequences in the opposite direction from expression (5' direction).
• Downstream: Sequences proceeding in the direction of expression within the transcription unit (3' direction).
• Primary transcript: The original, unmodified RNA product corresponding to a transcription unit.

RNA Polymerase

• Separates the two DNA strands in a transient bubble.
• Uses one strand (3' to 5') as a template to direct synthesis of complementary RNA (5' to 3').
• The length of the RNA-DNA hybrid within the bubble is approximately 8 to 9 base pairs.
• Transcription occurs via base pairing within this bubble of unpaired DNA strands.
• As the bubble moves, the DNA duplex reforms, displacing the RNA.

Transcription Stages

• Initiation
  • RNA polymerase binds to the promoter on the DNA, forming a closed complex.
  • RNA polymerase initiates transcription after opening the DNA duplex, forming a transcription bubble (open complex).
• Elongation
  • The transcription bubble moves along the DNA.
  • The RNA chain extends in the 5' to 3' direction by adding nucleotides to the 3' end.
  • Rate: 40-50 nucleotides/second, compared to 800 bp/second for DNA polymerase.
• Termination
  • Transcription stops, the DNA duplex reforms, and RNA polymerase dissociates at a terminator site.

Bacterial RNA Polymerase Holoenzyme

• Composed of six units:
  • α2ββ'ω core enzyme: Catalyzes transcription but cannot initiate it.
  • Sigma (σ) subunit: Required only for initiation (promoter recognition).
• Sigma factor alters the DNA-binding properties of RNA polymerase.
  • Reduces RNA polymerase affinity for general DNA.
  • Increases its affinity for promoters.
  • RNA polymerase affinity for general DNA isreduced by the sigma factor and its affinity for promoters is increased, due to its basic protein nature interacting with acidic nucleic acids.
• Approximately 13,000 RNA polymerase enzymes per E. coli cell.
• β and β’ subunits: Catalysis, most mass.
• α2 subunits: Scaffold, structure.
• ω subunit: Assembly regulatory.

Core Enzymes vs. Holoenzyme

• Core enzymes (without σ) have a general DNA binding affinity.
  • Can bind to DNA for up to 60 minutes.
  • Exhibit loose binding (less than 1 second).
  • Cannot distinguish promoters from other DNA sequences.
• Core enzymes with sigma factor (σ) have altered DNA affinity.
  • Reduces loose binding to less than 1 second.
  • Increases specific binding to promoter sequences by 1,000x.
  • Can bind to a promoter for several hours.

Promoter Recognition

• Nearly all RNA polymerase is bound to DNA; it is not free-floating and finds DNA by diffusion.
• Multiple models exist for finding promoter regions:
  • Sliding
  • Intersegment transport
  • Intradomain association and dissociation (hopping).

Complex Formation

• RNA polymerase binds to the promoter as a closed complex (DNA remains double-stranded).
• RNA polymerase (with sigma factor) then separates the DNA strands to form an open complex (transcription bubble).
• Incorporates up to nine nucleotides into RNA.
• Catalytic activity resides in the β and β’ subunits.

Ternary Complex and Abortive Initiation

• Ternary complex: Complex in initiation of transcription consisting of RNA polymerase, sigma factor, DNA, and a dinucleotide representing the first two bases in the RNA product.
• RNA polymerase may undergo cycles of abortive initiation, synthesizing short mRNA transcripts that are released before the transcription complex leaves the promoter.
• Sigma factor may be released from RNA polymerase when the nascent RNA chain reaches eight to nine bases in length.
• RNA polymerase initially contacts the region from -55 to +20.
• Promoter clearance time: How quickly the polymerase leaves the promoter for a new polymerase to start initiation. 1-2 seconds.

Conserved and Consensus Sequences

• Conserved sequence: A real sequence where the same individual bases or amino acids are always found at particular locations when comparing many examples of a nucleic acid or protein.
• Promoter: The site where RNA polymerase binds DNA, defined by short consensus sequences at specific locations.
• Consensus sequence: A model defined by aligning known nucleotide sequences of a regulatory region. The nucleotide occurring most often at each position is indicated.
  • Also applicable to protein sequences.

Promoter Consensus Sequences

• The promoter consensus sequences consist of:
  • A purine at the startpoint.
  • The hexamer TATAAT centered at -10 (Pribnow box).
  • The hexamer TTGACA centered at -35 (-35 box).
• Individual promoters usually differ from the consensus at one or more positions.
• Promoter efficiency can be affected by additional elements:
  • Extended -10 element on the upstream side.
  • Discriminator on the downstream side.
  • A-T rich regions far upstream called UP element. Interact with RNA polymerase and act as extra enhancers in highly expressed genes.

Sigma Factors

• E. coli has several sigma factors that recognize promoters with different consensus sequences.
  • σ⁷⁰ (RpoD): Housekeeping; regulates regular genes needed for normal growth (most common).
  • σ³² (RpoH): Heat shock; regulates genes that help cells survive high temperatures.
  • σ⁵⁴ (RpoN): Nitrogen limitation; regulates genes involved in nitrogen metabolism.
  • σ²⁴ (RpoE): Extracytoplasmic stress; responds to misfolded proteins in the membrane.

Mutations and Promoter Efficiency

• Down mutations (decrease promoter efficiency) decrease conformance to the consensus sequences.
• Up mutations (increase promoter efficiency) increase conformance to the consensus sequence.
• Mutations in the -35 sequence can affect the initial binding of RNA polymerase.
• Mutations in the -10 sequence usually affect the melting reaction that converts a closed complex to an open complex.
  • TATAAT has just A and T, min amount of energy to melt
  • Also ensure directionality
• A perfect consensus decreases the transition into elongation.

Sigma 70

• σ70 changes its structure to expose its DNA-binding regions when it associates with the core enzyme.
  • Otherwise, it does not interact with the DNA.
  • This helps to regulate gene expression at a basal level.
• σ70 binds both the -35 and -10 sequences.

Supercoiling

• Negative supercoiling increases the efficiency of some promoters by assisting the melting reaction.
• Transcription generates:
  • Positive supercoils ahead of the enzyme.
  • Negative supercoils behind it.
• Negative supercoils must be introduced by gyrase.
• Topoisomerase relaxes negative supercoils.

Bacterial Transcription Termination

Two types of termination sites:

• Recognition of the terminator sequence in DNA by a sequence-specific factor that works with RNA polymerase.
• The formation of a hairpin structure in the RNA product.

Intrinsic Terminators

• Consist of a G-C-rich hairpin in the RNA product followed by a U-rich region where termination occurs.
• Causes an approx. 60-second delay.
• The string of U’s and the RNA-DNA hybrid (rU-dA) is the weakest association.
• During the delay, RNA can dissociate from DNA, thus terminating transcription
  • But this alone ranges from 2-90% efficiency
  • Other mechanisms needed

Rho Factor

• Rho factor is a terminator protein that binds to a rut site on a nascent RNA.
• rut (rho utilization site): RNA sequence recognized by the rho termination factor.
• RNA polymerase pauses at a hairpin structure in the 3' UTR.
• Rho factor tracks along the RNA to release it from the RNA-DNA hybrid structure at the RNA polymerase.
• Rho is a helicase (requires ATP).
• A rut site has a sequence rich in C and poor in G preceding the actual site of termination.
  • The sequence corresponds to the 3′ end of the RNA.

Rho structure

• Rho has an N-terminal RNA binding domain and a C-terminal ATPase domain
• Member of the family of hexameric ATP- dependent helicases
• Each subunit has: an RNA- binding domain and an ATP hydrolysis domain
• Rho uses its helicase activity to:
  • unwind the duplex structure
  • help pry off the RNA

Bacterial mRNA Cycle

• Transcription and translation occur simultaneously in bacteria (coupled transcription/translation).
  • Ribosomes begin translating an mRNA before its synthesis is complete.
• Bacterial mRNA is unstable, with a half-life of only a few minutes.

mRNA Terminology

• Nascent RNA: An RNA chain still being synthesized, with its 3' end paired with DNA where RNA polymerase is elongating.
• Monocistronic mRNA: mRNA that encodes one polypeptide.
• Bacterial mRNA may be polycistronic, having several coding regions representing different cistrons.
• 5' UTR: The untranslated sequence upstream from the coding region of an mRNA.
• 3' UTR: The untranslated sequence downstream from the coding region of an mRNA.
• Intercistronic region: In a polycistronic mRNA, the distance between the termination codon of one cistron and the initiation codon of the next cistron.