Transcription and Gene Expression: Prokaryotic and Eukaryotic Mechanisms

Gene Expression: An Overview

The Central Dogma of Molecular Biology

Gene expression is the process by which information encoded in DNA is used to produce a functional product, typically a protein. This process is fundamental to all living organisms and is summarized by the central dogma:

• Transcription: DNA is transcribed into RNA by RNA polymerase.

• RNA Processing: In eukaryotes, the primary RNA transcript (pre-mRNA) undergoes modifications such as splicing, capping, and polyadenylation.

• Translation: The processed mRNA is translated into protein by ribosomes.

Example: The gene for hemoglobin is transcribed into mRNA, which is then translated into the hemoglobin protein.

Transcription in Prokaryotes

RNA Polymerase and Promoters

Transcription in prokaryotes is carried out by a single type of RNA polymerase, which recognizes specific DNA sequences called promoters to initiate transcription.

• RNA Polymerase: Composed of a core enzyme and a sigma (σ) subunit. The sigma subunit confers promoter specificity.

• Promoter Elements: Key regions include the -35 and -10 elements upstream of the transcription start site (+1).

• Consensus Sequences: Typical sequences at -35 (TTGACA) and -10 (TATAAT) positions.

Example: The lac operon promoter in E. coli contains -35 and -10 elements recognized by the σ70 subunit.

Stages of Prokaryotic Transcription

• Initiation: RNA polymerase holoenzyme binds to the promoter, DNA unwinds at the -10 region, and transcription begins at +1.

• Elongation: The sigma subunit dissociates, and the core enzyme synthesizes RNA. About 18 base pairs of DNA are unwound at a time, and a short RNA-DNA hybrid is maintained.

• Termination: Occurs at defined sequences downstream of the coding region. Two main types:

  • Intrinsic (Rho-independent): Formation of a GC-rich hairpin loop in the RNA followed by a string of uracils causes RNA polymerase to dissociate.

  • Rho-dependent: The Rho protein binds to the rut site on RNA and moves toward the polymerase, causing dissociation when it catches up.

Equation: $\text{DNA (template)} \xrightarrow{\text{RNA polymerase}} \text{RNA}$

Concurrent Transcription and Translation

In prokaryotes, transcription and translation are often coupled, meaning ribosomes begin translating mRNA while it is still being synthesized.

Transcription in Eukaryotes

RNA Polymerases

Eukaryotes have three main RNA polymerases, each responsible for transcribing different types of genes:

Polymerase	Location	Products
RNA Polymerase I	Nucleolus	Ribosomal RNAs (except 5S rRNA)
RNA Polymerase II	Nucleus	Pre-mRNAs (protein-coding genes)
RNA Polymerase III	Nucleus	tRNAs, 5S rRNA, other small RNAs

Structure of Eukaryotic mRNA

• 5' Cap (7-methylguanosine): Added to the 5' end for stability and initiation of translation.

• 5' Untranslated Region (5' UTR): Regulatory region upstream of the coding sequence.

• Coding Sequence (CDS): Region that encodes the protein (from start codon AUG to stop codon UAA, UGA, or UAG).

• 3' Untranslated Region (3' UTR): Regulatory region downstream of the coding sequence.

• Poly(A) Tail: String of adenines added to the 3' end, promoting stability and translation.

Promoter Elements and General Transcription Factors

Promoters for RNA Polymerase II (Pol II) contain several consensus elements:

• TATA Box: Located around -25, recognized by TATA-binding protein (TBP).

• CAAT Box: Located around -80, involved in promoter efficiency.

• GC-rich Box: Located upstream, involved in regulation.

General transcription factors (GTFs) are required for Pol II transcription initiation:

• TFIID: Contains TBP, binds TATA box.

• TFIIB, TFIIF, TFIIE, TFIIH: Assemble at the promoter to form the pre-initiation complex (PIC).

• TFIIH: Unwinds DNA and phosphorylates RNA Pol II to initiate transcription.

Stages of Eukaryotic Transcription (Pol II)

• Initiation: GTFs and RNA Pol II assemble at the promoter to form the PIC. DNA is unwound, and transcription begins at the transcription start site (TSS).

• Elongation: RNA Pol II synthesizes RNA, and most GTFs dissociate.

• Termination: (To be covered next time) Involves cleavage of the transcript and addition of the poly(A) tail.

Mutational Analysis of Promoters

Functional elements of promoters can be identified by introducing mutations and measuring effects on gene expression. For example, mutating the TATA box often reduces transcription levels.

Experimental Methods for Studying Transcription

Electrophoretic Mobility Shift Assay (EMSA)

EMSA, or band shift assay, is used to detect protein-DNA interactions in vitro. A labeled DNA fragment is incubated with a protein extract; if the protein binds, the DNA-protein complex migrates more slowly during gel electrophoresis.

• Purpose: To confirm and study the binding of transcription factors or other proteins to specific DNA sequences.

Chromatin Immunoprecipitation (ChIP)

(To be covered next time) ChIP is used to study protein-DNA interactions in vivo by crosslinking proteins to DNA, immunoprecipitating the complex, and identifying the bound DNA sequences.

Key Terms and Definitions

• Gene Expression: The process by which information from a gene is used to synthesize a functional gene product.

• Transcription: Synthesis of RNA from a DNA template.

• Translation: Synthesis of protein from an mRNA template.

• Promoter: DNA sequence where RNA polymerase binds to initiate transcription.

• RNA Polymerase: Enzyme that synthesizes RNA from a DNA template.

• General Transcription Factors: Proteins required for the initiation of transcription by RNA Pol II.

• Pre-initiation Complex (PIC): Assembly of GTFs and RNA Pol II at the promoter.

• Intrinsic Termination: Termination of transcription due to RNA secondary structure (hairpin loop).

• Rho-dependent Termination: Termination requiring the Rho protein.

Summary Table: Comparison of Prokaryotic and Eukaryotic Transcription

Feature	Prokaryotes	Eukaryotes
Location	Cytoplasm	Nucleus
RNA Polymerases	One	Three (I, II, III)
Promoter Elements	-10, -35	TATA, CAAT, GC boxes
Initiation Factors	Sigma subunit	General transcription factors
RNA Processing	Minimal	Capping, splicing, polyadenylation
Coupling of Transcription & Translation	Yes	No

Additional info: Some details about eukaryotic transcription termination and chromatin immunoprecipitation are noted as 'to be covered next time' in the original notes.