Crick’s Central Dogma and Gene Expression

  • Background: Watson & Crick described the DNA double helix in 1953.
    • In 1956, Crick proposed the Central Dogma to explain the flow of genetic information: DNA → RNA → Protein.
    • Central Dogma today is a one-way process of gene expression: “Once information has passed into protein, it cannot get out again.”
  • Key idea: Proteins are the final products of gene expression and determine many cellular properties and capabilities.

Proteins and Gene Expression (Overview)

  • Proteins Rule Over All (summary ideas):
    • Proteins control: Structure, Function, Development, Reproduction.
    • Proteins affect: Behavior, Physiology, Survival.
  • Source reference: https://twitter.com/MarkScherz/status/1144686897630994433

How We Make Proteins: From Genes to Polypeptides

  • Proteins are polypeptide strings of amino acids.
  • Genes (functional units of DNA) code for the sequence of amino acids in a protein.
  • Transcription: synthesis of an RNA copy (mRNA) of a DNA template.
  • Translation: synthesis of proteins by converting the mRNA “code” into amino acids in a polypeptide.

A Transcript: Similar Language, Different Form

  • Transcribing = making a transcript (RNA copy).
  • Translation = interpretation of meaning of a text and producing an equivalent text in another language (metaphor used to describe converting RNA to protein).
  • Transcript uses RNA; message is carried in mRNA.

Crick’s Central Dogma in Detail

  • Recall the discovery: DNA → RNA → Protein.
  • Nature of the process: predominantly unidirectional in gene expression.
  • Quote: “Once information has passed into protein, it cannot get out again.”

Genes: Transcription, Translation, and Genomic Elements

  • Not all genes are transcribed or translated; introns are examples that may not be transcribed or translated.
  • A gene often includes: promoter, transcribed sequence, and regulatory elements.
  • Differences in processes and enzymes between prokaryotes and eukaryotes are important for understanding test questions.

RNA and Its Roles in Gene Expression

  • Messenger RNA (mRNA): translated into protein.
  • Ribosomal RNA (rRNA): component of ribosomes (where proteins are made).
  • Transfer RNA (tRNA): brings amino acids to ribosomes.
  • Small nuclear RNA (snRNA): splicing (exclusion of introns) in eukaryotes.
  • Micro RNA (miRNA): blocks translation of targeted genes in eukaryotes.

Transcription Goals: RNA Types, Polymerase, and Differences

  • RNA trends and types: overview of RNA species and their roles.
  • RNA polymerase: initiation, elongation, termination.
  • Differences between Eukaryotes and Prokaryotes are important for tests.

Transcription Terminology and Process Similarities to DNA Synthesis

  • Synonyms and terminology:
    • RNA-like strand, coding strand, sense strand, nontemplate strand.
  • Transcription’s basic property:
    • Synthesized mRNA has the same polarity and sequence as the nontemplate strand (with U replacing T).
    • The nontemplate strand is RNA-like.
  • Nucleotide usage in transcription:
    • Uses NTPs (not dNTPs); UTP replaces dTTP; no primer is needed.
    • Transcription is more error-prone than DNA synthesis.
  • Directionality:
    • Only one strand is transcribed.
    • Transcription proceeds in a similar manner to DNA synthesis: from 5′ to 3′ on the growing RNA chain.

Prokaryotic Phases of Transcription

1) Initiation

  • Promoter is analogous to a replicator sequence.
  • No primer required.
  • Sigma factor subunit (σ) helps initiate.
  • Short synthesis of ~8–10 nucleotides before promoter clearance.
    2) Elongation
  • Sigma is usually lost; holoenzyme transitions to elongation.
  • RNA polymerase holoenzyme moves along DNA to form a transcription bubble.
  • RNA synthesis proceeds 5′ to 3′ direction.
  • Once an elongation complex moves past the promoter, another holoenzyme can initiate at the promoter again.
    3) Termination
  • Transcription ends at terminator sequences:
    • Intrinsic (Rho-independent) terminators form hairpin structures in the RNA to terminate transcription.
    • Extrinsic (Rho-dependent) terminators involve the Rho helicase (ρ) and a rut site; it causes termination by disengaging the holoenzyme from DNA.

Prokaryotic Initiation Details

  • Sigma factor (σ) binds to the promoter as part of the RNA polymerase holoenzyme.
  • It aligns the first two NTPs and catalyzes the phosphodiester bond formation.
  • It synthesizes a short RNA stretch (~8–10 NTPs) and then detaches, allowing growth to continue with the core RNA polymerase.

Prokaryotic Elongation Details

  • After sigma dismisses, the elongation phase proceeds with the holoenzyme.
  • The polymerase unwinds DNA to create a transcription bubble.
  • The mRNA chain is extended in the 5′ to 3′ direction.
  • New RNA polymerase molecules can begin transcription as others move off the promoter.

Termination Mechanisms in Prokaryotes

  • Intrinsic (Rho-independent) terminators:
    • Hairpin structures in RNA prematurely halt RNA polymerase.
  • Extrinsic (Rho-dependent) terminators:
    • Rho protein binds at the rut site on the RNA and travels toward the 3′ end.
    • When RNA polymerase encounters the terminator, it pauses; rho catches up and, via helicase activity, unwinds the DNA-RNA hybrid to end transcription.

Prokaryotic Termination Illustrations

  • Example sequences and hairpin formation show termination hairpins forming in RNA and RNA polymerase stopping at the hairpin.
  • Visuals depict RNA, DNA, and terminator interactions during termination.

Upstream vs Downstream Orientation in Transcription

  • Upstream vs downstream refers to the direction RNA polymerase moves during transcription.
  • The transcription start site is considered the 5′ end of the nascent RNA; upstream is negative relative to start, downstream is positive.

mRNA Molecules: Structure and Translation Coupling

  • All mRNAs have three main parts: 5′ leader, coding sequence, and 3′ trailer.
  • Prokaryotes can be polycistronic (operons) where a single mRNA encodes multiple genes.
    • In bacteria, operons lead to mRNAs that contain multiple genes and can be translated while transcription is still ongoing (coupled transcription-translation).
  • Eukaryotic mRNAs are monocistronic (one gene per mRNA).

Transcription in Eukaryotes: Key Differences and Organization

  • Transcription occurs in the nucleus; translation occurs in the cytoplasm.
  • Chromatin must be uncoiled to expose DNA to RNA polymerase.
  • Eukaryotes have much more DNA and more genes than prokaryotes (roughly 700× more DNA and about 10× more genes).
  • Initiation involves a more complex and highly specific set of proteins.
  • The initial product is pre-mRNA, not yet mature mRNA; RNA processing occurs before export to cytoplasm.

RNA Polymerases: Prokaryotes vs Eukaryotes

  • Prokaryotes:
    • A single RNA polymerase transcribes mRNA, tRNA, and rRNA, and also helps unwind DNA.
  • Eukaryotes:
    • RNA Polymerase I: rRNA synthesis (except some classes).
    • RNA Polymerase II: mRNA, miRNA, some snRNA; also involved in transcription of many protein-coding genes.
    • RNA Polymerase III: tRNA, snRNA, some rRNA.
    • In eukaryotes, Pols do not unwind DNA themselves; they require accessory proteins.

Eukaryotic Nuclear Organization and RNA Types

  • Nucleolus: location of rRNA synthesis.
  • Nucleoplasm: the fluid inside the nucleus where most transcription occurs.
  • Eukaryotic RNA processing involves capping, splicing of introns, and polyadenylation to produce mature mRNA.

Cis-acting vs Trans-acting Elements: Regulation of Transcription

  • cis-acting elements:
    • DNA sequences on the same molecule as the gene that regulate transcription by binding DNA-binding proteins.
    • Examples:
    • Promoter: close to the transcription start site; binds RNA polymerase and transcriptional machinery to start transcription.
    • Core promoter: includes elements like Inr (initiator) and the TATA box.
    • Promoter-proximal elements and enhancers: bind activators to maximize or repress transcription initiation.
    • Note: Some cis elements can function even if reversed or moved far from the gene.
  • trans-acting elements:
    • Factors encoded elsewhere in the genome that act on target genes by interacting with cis elements.
    • Usually transcription factors (and regulatory RNAs) that regulate transcription.
    • All three eukaryotic RNA polymerases require transcription factors (GTFs) to function.

Preinitiation Complex (PIC) and GTFs

  • General Transcription Factors (GTFs) must bind to the promoter first.
  • RNA Polymerase II can then be recruited to the promoter to start transcription.

Prokaryotic vs Eukaryotic Transcription: Key Differences Recap

  • Location:
    • Prokaryotes: transcription and translation occur in the cytoplasm; transcription is coupled to translation; mRNA is often polycistronic.
    • Eukaryotes: transcription occurs in the nucleus; mRNA processing occurs in nucleus; translation occurs in cytoplasm; mRNA is monocistronic.
  • Chromatin and promoters:
    • Eukaryotic promoters require a larger set of transcription factors and chromatin remodeling to access DNA.
  • Exceptions:
    • Some organisms (e.g., Caenorhabditis elegans) can have polycistronic mRNA but are still primarily monocistronic in many contexts.
  • Evolutionary note: Archaea share several transcriptional features with eukaryotes, more so than with bacteria, reflecting evolutionary relationships.

Practice Quiz: Transcription (Sample Question and Answer)

  • Question: The coding sequence for gene F is read from left to right on the figure below. The coding sequence for gene G is read from right to left. Which strand of DNA (top or bottom) serves as the template for transcription of each gene?
    • 5′ 5′ 3′ 3′
    • Gene F Gene G
  • Answer options summarized (from notes):
    • A. Gene F: top; Gene G: top
    • B. Gene F: bottom; Gene G: bottom
    • C. Gene F: bottom; Gene G: top ← Correct answer given
    • D. Gene F: top; Gene G: bottom
  • Rationale: Coding sequence corresponds to the mRNA sequence (with U for T); mRNA is synthesized 5′ to 3′. The gene orientation determines which strand serves as the template; in the provided solution, Gene F uses the bottom strand as template and Gene G uses the top strand.

Practice Quiz Solution (Summary)

  • Correct Answer: Gene F uses bottom strand as template; Gene G uses top strand as template (Option C).

Quick Reference: Key Terms and Concepts to Remember

  • Central Dogma: DNA → RNA → Protein.
  • Transcription produces mRNA; Translation produces protein.
  • Prokaryotic transcription phases: Initiation, Elongation, Termination.
  • Terminators:
    • Intrinsic (Rho-independent) hairpin in RNA.
    • Extrinsic (Rho-dependent) requires ρ protein and rut site.
  • mRNA structure: 5′ leader, coding sequence, 3′ trailer; polycistronic in bacteria; monocistronic in eukaryotes.
  • Eukaryotic transcription involves: nucleus, chromatin remodeling, three RNA polymerases, and a pre-mRNA product that must be processed.
  • cis-acting elements regulate transcription on the same DNA molecule; promoters and enhancers.
  • trans-acting elements regulate transcription via diffusible factors (transcription factors); GTFs are required for RNA polymerases in eukaryotes.
  • Directionality and orientation cues: transcription proceeds 5′ to 3′; upstream is before start site; downstream is after start site.
  • Important numerical references:
    • Initiation length: ~8–10 nucleotides (8−10  nt8{-}10\ \,\text{nt}).
    • Elongation rate: 40−50 nt/s40{-}50\,\text{nt/s} at 37∘C37^{\circ}\mathrm{C}.
    • Hairpin and terminator structures conceptualize termination (no fixed numeric rate given).
    • Prokaryotes: single RNA polymerase for mRNA, tRNA, rRNA; Eukaryotes: Pol I, II, III with specialized roles.
    • Prokaryotic vs Eukaryotic genome size: eukaryotes have roughly 700×700\times more DNA and ∼10×\sim 10\times more genes than prokaryotes.

Notes: Some slides reference figures or slide numbers (e.g., Fig. 13.14, Fig. 13.13); these are mentioned here to preserve context from the transcript. If you encounter these in lectures or exams, they correspond to intrinsic/extrinsic termination and the rho-dependent mechanism, respectively.