RNA Transcription Notes

Transcription

Overview

  • Transcription is the conversion of genetic information from DNA to RNA.
  • Sense Strand: Same as RNA (coding strand/non-template strand).
  • Antisense Strand: Complementary to RNA (non-coding strand/template strand).

DNA Strands

  • The coding strand has the same base sequence as the RNA transcript (U instead of T).
  • The template strand serves as a template for RNA synthesis.
  • The coding strand can be located on either strand of a chromosome.

RNA vs DNA

  • Nitrogenous Bases: RNA contains uracil (U) instead of thymine (T).
  • Sugar: RNA contains ribose, while DNA contains deoxyribose.
  • Strand Structure: RNA is typically single-stranded.
  • Chargaff's Rules: Do not apply to RNA (A ≠ U, C ≠ G).

RNA Structure and Function

  • Single-stranded RNA can form three-dimensional structures by looping and interacting with itself.
  • Cellular Transcriptome: The sum of all RNA molecules produced in a cell under specific conditions.

Types of RNA

Messenger RNA (mRNA)
  • Carries genetic information from DNA for protein synthesis.
  • Constitutes approximately 5% of cellular RNA.
  • Varies in size (e.g., 500-6000 nucleotides in E. coli).
  • Eukaryotic mRNA has unique modifications.
  • Prokaryotic mRNAs are often polycistronic while eukaryotic mRNA is typically monocistronic.
  • Eukaryotic mRNA undergoes modifications such as:
    • Capping (7-methylguanosine at the 5' end).
    • Splicing (removal of introns).
    • Addition of a poly(A) tail.
  • Introns: Noncoding sections spliced out.
  • Exons: Coding sections.
Transfer RNA (tRNA)
  • Transports amino acids to ribosomes for protein assembly.
  • Comprises about 15% of cellular RNA.
  • Average length is 75 nucleotides.
  • Each tRNA binds to a specific amino acid.
  • Cells have at least one tRNA type for each of the 20 common amino acids.
  • Has a cloverleaf-like three-dimensional structure with five arms.
  • Contains modified bases (e.g., pseudouridine, 4-thiouridine, 1-methylguanosine, dihydrouridine).
Ribosomal RNA (rRNA)
  • Most abundant RNA form (approximately 80% of total RNA).
  • Has a complex secondary structure, giving it catalytic activity.
  • Ribosomes synthesize proteins from mRNA.
  • Prokaryotic and eukaryotic ribosomes are similar but differ in size and chemical composition.
  • Both ribosome types consist of two subunits, referred to by their Svedberg (S) unit values.
MicroRNA (miRNA)
  • Short, single-stranded RNAs (20-22 nucleotides).
  • Cleaved from hairpin-shaped precursors.
  • Function in gene regulation in eukaryotes.
  • Inhibit gene expression by binding to target mRNA and inhibiting translation.
Small Nuclear RNA (snRNA)
  • Small RNA molecules (90-300 nucleotides).
  • Complexed with proteins to form small nuclear ribonucleoprotein particles (snRNPs or snurps).
  • Involved in splicing and other RNA processing activities.

E. coli RNA Polymerase

  • Complex, multimeric protein.
  • Molecular weight of ~480,000.
  • Consists of five polypeptides, four of which are distinct.
  • Holoenzyme composition: α2ββ′σ\alpha_2\beta\beta'\sigma
    • α subunits: involved in the assembly of the tetrameric core (α2ββ′\alpha_2\beta\beta').
    • β subunit: contains the ribonucleoside triphosphate binding site.
    • β’ subunit: harbors the DNA template-binding region.
    • σ factor: involved only in the initiation of transcription; recognizes and binds RNA polymerase to promoter sites.
  • Core enzyme (α2ββ′\alpha_2\beta\beta') catalyzes RNA synthesis from DNA templates in vitro but initiates at random sites.
  • Holoenzyme (with σ) initiates RNA chains in vitro only at sites used in vivo.

Steps of Transcription

  1. Initiation (requires sigma factor with the core enzyme).
  2. Elongation (performed by core enzyme).
  3. Termination (rho-dependent or rho-independent).

Requirements of Transcription

  1. A promoter: a sequence upstream of the start of the gene encoding the RNA.
    • RNA polymerase interacts with the promoter.
    • Orients RNA polymerase to start transcribing at the beginning of the gene, ensuring consistent initiation.
    • A gene with its promoter is an independent unit.
  2. The RNA-coding sequence: the DNA sequence transcribed by RNA polymerase into the RNA transcript.
  3. A terminator: specifying where transcription stops.

Promoter Region

  • RNA polymerase binds to specific promoter sequences on DNA.
  • In E. coli, binding occurs within a region from -70 bp to +30 bp relative to the transcription start site.
  • The first base of the RNA molecule is +1; preceding bases are negative numbers.
  • Important interaction sites for the sigma subunit are centered at -10 and -35.
  • These sites contain consensus sequences.
    • -35 box consensus sequence: 5’-TTGACA-3’.
    • -10 box (Pribnow box) consensus sequence: 5’-TATAAT-3’.
  • Efficiency of RNA polymerase binding and transcription initiation depends on these sequences, their spacing, and their distance from the transcription start site.

RNA Chain Initiation

  • Polymerase holoenzyme (with sigma factor) binds to the promoter (closed promoter complex).
  • Holoenzyme untwists DNA in the -10 region (open promoter complex).
  • RNA polymerase is oriented to begin transcription at the correct nucleotide.
  • RNA polymerase contacts about 75 bp of DNA from -55 to +20.
  • Sigma subunit dissociates as the polymerase enters the elongation phase.
  • Short chains of 2-9 ribonucleotides are synthesized and released during initiation.
  • Abortive synthesis stops once chains of 10 or more ribonucleotides have been synthesized.
  • Conformational change converts the complex to the elongation form (promoter clearance).

RNA Chain Elongation

  • Catalyzed by the RNA polymerase core enzyme (after sigma subunit release).
  • Covalent extension of RNA chains occurs within the transcription bubble (a locally unwound DNA segment).
  • RNA polymerase has DNA unwinding and rewinding activities.
  • Average transcription bubble length in E. coli is 18 nucleotide pairs.
  • About 40 ribonucleotides are incorporated per second.
  • The nascent RNA chain is displaced from the DNA template strand.
  • Transient base-pairing between the growing chain and template strand is very short (perhaps only three base pairs).
  • The stability of the transcription complex is maintained by the binding of DNA and RNA to RNA polymerase.

RNA Chain Termination

  • Occurs when RNA polymerase encounters a termination signal.
  • The transcription complex dissociates, releasing the nascent RNA molecule.
  • Two types of terminators in E. coli:
    • Rho-dependent terminators (require rho (ρ) protein).
    • Rho-independent terminators (do not require rho).
Rho-independent Termination
  • Contain a GC-rich region followed by six or more AT base pairs (A’s in the template strand).
  • The GC-rich region contains inverted repeats.
  • Inverted repeat (IR): a single-stranded sequence followed by its reverse complement.
  • Inverted repeat regions produce single-stranded RNA sequences that can base-pair and form hairpin structures.
  • RNA hairpin structures retard RNA polymerase movement, causing pauses in chain extension.
  • Polymerase complex undergoes a conformational change (isomerization) at the terminator site.
  • Hairpin disrupts RNA-DNA hybrid or RNA-polymerase interactions, resulting in isomerization.
  • Unstable A-U hybrid region at the 3’ end leads to RNA dissociation and termination.
Rho-dependent Termination
  • Lack repeated A residues in the template strand.
  • Contain:
    • A 50–90 nucleotide-pair sequence upstream from inverted repeats, producing an RNA strand rich in C’s but few G’s (no hairpins).
    • A CA-rich rho protein binding site (rut element) near the 3’ end of the transcript.
  • Rho protein associates with RNA at rut sites and migrates in the 5’-3’ direction to the paused transcription complex.
  • Contributes to release of the RNA transcript.
  • Rho protein has ATP-dependent RNA-DNA helicase activity, which translocates the protein along the RNA.
  • Helicase unwinds the RNA-DNA helix; ATP is hydrolyzed during the termination process.

Eukaryotic RNA Polymerases

  • Eukaryotes have at least three RNA polymerases (I, II, and III).
  • More complex than E. coli RNA polymerase and require transcription factors.
    • RNA polymerase I: located in the nucleolus; synthesizes most ribosomal RNAs (except 5S rRNA).
    • RNA polymerase II: transcribes nuclear genes that encode proteins and synthesizes other primary transcripts.
    • RNA polymerase III: catalyzes the synthesis of tRNA, 5S rRNA, and small nuclear RNAs.

Eukaryotic Upstream Sequences

  • RNA polymerase II promoters consist of short conserved elements upstream from the transcription start-point.
  • TATA box: consensus sequence TATAAAA, centered at about position -30; important for positioning the transcription start-point.
  • CAAT box: consensus sequence GGCCAATCT, usually near position -80.
  • GC box: consensus sequence GGGCGG.
  • Octamer box: consensus sequence ATTTGCAT.
  • RNA polymerase II promoters contain some, but not all, of these components.

Prokaryotic and Eukaryotic mRNA

  • In bacteria, mRNA does not require processing before translation; transcription and translation are coupled.
  • In eukaryotes, the primary RNA transcript is a precursor-mRNA (pre-mRNA).
  • Pre-mRNA is processed in the nucleus by:
    • Addition of a 5’ cap.
    • Addition of a 3’ poly(A) tail.
    • Removal of introns (splicing).
  • Translation occurs only after mRNA is transported to the cytoplasm.

Eukaryotic mRNA Processing

  • Three major modifications occur prior to transport to the cytoplasm:
    1. 7-Methyl guanosine caps are added to the 5’ ends.
    2. Poly(A) tails are added to the 3’ ends.
    3. Intron sequences are spliced out.
  • 5’ cap: 7-methyl guanosine residue joined to the initial nucleoside by a 5’-5’ phosphate linkage.
  • 3’ poly(A) tail: a polyadenosine tract 20 to 200 nucleotides long.