Transcription and RNA Processing: Key Concepts in Genetic Analysis

Transcription and RNA Processing

Overview

This unit explores the molecular mechanisms of transcription and RNA processing, focusing on the central dogma of biology, the roles of RNA polymerases, and the differences between prokaryotic and eukaryotic systems. Understanding these processes is fundamental to the study of gene expression in genetics.

Central Dogma of Biology

Definition and Importance

• Central Dogma: The flow of genetic information in cells proceeds from DNA to RNA to Protein.

• Transcription: The process of copying genetic information from DNA to RNA.

• Translation: The process by which ribosomes synthesize proteins using the information encoded in mRNA.

Example: In both prokaryotes and eukaryotes, DNA is transcribed into messenger RNA (mRNA), which is then translated into proteins by ribosomes.

Transcription: The First Step in Gene Expression

Definition and Enzyme Involved

• Transcription is the synthesis of RNA from a DNA template.

• The enzyme responsible is RNA polymerase.

• In prokaryotes, there is a single RNA polymerase for all types of RNA.

• In eukaryotes, multiple RNA polymerases exist, each with specialized functions.

DNA Strands in Transcription

Coding vs. Template Strand

• The template strand (3' to 5') is read by RNA polymerase to synthesize RNA in the 5' to 3' direction.

• The coding strand (5' to 3') has the same sequence as the RNA (except T is replaced by U in RNA).

RNA Polymerases

Prokaryotic vs. Eukaryotic RNA Polymerases

• Prokaryotes: One RNA polymerase transcribes all types of RNA.

• Eukaryotes: Three main RNA polymerases, each transcribing different classes of genes.

Form	Product	Location
I	rRNA	Nucleolus
II	mRNA, snRNA	Nucleoplasm
III	5S rRNA, tRNA	Nucleoplasm

Transcription and Translation: Prokaryotes vs. Eukaryotes

Key Differences

• Prokaryotes: Transcription and translation are coupled; both occur in the cytoplasm.

• Eukaryotes: Transcription occurs in the nucleus; translation occurs in the cytoplasm. RNA processing is required before translation.

Example: In prokaryotes, ribosomes can begin translating mRNA while it is still being transcribed. In eukaryotes, mRNA must be processed and exported from the nucleus before translation.

Promoters and Initiation of Transcription

Definition and Function

• Promoter: A DNA sequence upstream of the transcription start site that signals RNA polymerase where to begin transcription.

• In prokaryotes, promoters contain consensus sequences at -35 and -10 (Pribnow box) positions.

• In eukaryotes, promoters are recognized by general transcription factors (GTFs) and RNA polymerase II.

Structure of E. coli RNA Polymerase

Subunit Composition

• Composed of multiple subunits: two α, one β, one β', one ω, and a σ (sigma) factor.

• The core enzyme (α2ββ'ω) is responsible for RNA synthesis.

• The sigma factor provides promoter specificity and is required for initiation.

Example: The σ70 factor in E. coli recognizes promoters for housekeeping genes.

Stages of Transcription in Prokaryotes

1. Initiation

• RNA polymerase holoenzyme binds to the promoter.

• DNA strands are separated to form an open complex.

• Transcription begins at the +1 site.

2. Elongation

• RNA polymerase moves along the template strand, synthesizing RNA in the 5' to 3' direction.

• The transcription bubble is maintained as DNA unwinds ahead and rewinds behind the polymerase.

3. Termination

• Transcription ends by either intrinsic (rho-independent) or rho-dependent mechanisms.

Termination Mechanisms in Prokaryotes

Intrinsic (Rho-Independent) Termination

• Relies on a GC-rich hairpin structure in the RNA followed by a series of uracils.

• The hairpin causes RNA polymerase to pause and dissociate from the DNA.

Rho-Dependent Termination

• Requires the Rho protein, which binds to the rut site on RNA.

• Rho uses ATPase and helicase activity to separate the RNA from the DNA template, releasing the transcript.

Transcription in Eukaryotes

RNA Polymerases and Promoters

• Three RNA polymerases (I, II, III) transcribe different gene classes.

• Promoters are recognized by general transcription factors (GTFs) such as TFIIA, TFIIB, TFIID, TBP, etc.

• The preinitiation complex positions RNA polymerase II at the transcription start site.

RNA Processing in Eukaryotes

1. 5' Capping

• A 7-methylguanosine cap is added to the 5' end of the nascent RNA.

• Functions: Protects RNA from degradation and is required for translation initiation.

2. Splicing

• Removal of non-coding introns and joining of exons.

• Splicing is carried out by the spliceosome, a complex of small nuclear RNAs (snRNAs) and proteins (snRNPs or "snurps").

• Conserved sequences at intron-exon boundaries (GU at 5' end, AG at 3' end) are recognized by the splicing machinery.

3. 3' Polyadenylation

• After transcription, a poly(A) tail (150-200 adenines) is added to the 3' end of the RNA.

• Functions: Enhances stability and export of mRNA from the nucleus.

Termination of Transcription in Eukaryotes

Models of Termination

• Allosteric Model: RNA polymerase II undergoes a conformational change after passing the polyadenylation signal, leading to termination.

• Torpedo Model: An exonuclease degrades the RNA downstream of the cleavage site, causing RNA polymerase II to dissociate.

Summary Table: Prokaryotic vs. Eukaryotic Transcription

Feature	Prokaryotes	Eukaryotes
Location	Cytoplasm	Nucleus
RNA Polymerases	One	Three (I, II, III)
RNA Processing	Minimal	Extensive (capping, splicing, polyadenylation)
Coupling of Transcription & Translation	Yes	No

Key Terms

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

• Promoter: DNA sequence that directs RNA polymerase to the correct initiation site.

• Spliceosome: Ribonucleoprotein complex responsible for removing introns from pre-mRNA.

• snRNPs (snurps): Small nuclear ribonucleoproteins, essential components of the spliceosome.