Transcription and RNA Processing: From Genes to Functional mRNA

Transcription and RNA Processing

Introduction to Transcription

Transcription is the process by which the genetic information encoded in DNA is copied into RNA. This is a fundamental step in gene expression, allowing cells to produce proteins based on their genetic instructions.

• Human chromosomes contain approximately 3.1 billion base pairs and about 46,000 genes.

• Less than 1.5% of human DNA codes for proteins; the rest includes regulatory sequences and non-coding regions.

• Genes are identified by their specific sequences and regulatory elements.

What is a Gene?

A gene is a short stretch of DNA on a chromosome that contains the information necessary to produce a functional product, typically a protein.

• Genes have two main parts:

  • Regulatory region (upstream): Controls gene expression and contains binding sites for RNA polymerase and other factors.

  • Coding region (downstream): Contains the sequence that is transcribed and translated into protein.

• Gene expression occurs in two stages:

  1. Transcription: DNA is copied into RNA.

  2. Translation: RNA is used to synthesize protein.

Transcription

Mechanism of Transcription

Transcription is similar to DNA replication but with key differences:

• Only one of the two DNA strands is copied (the template strand).

• The product is RNA, not DNA.

• RNA polymerase synthesizes RNA without the need for a primer.

Example:

• DNA template: 3'-ATCGGCAGGACCTTAAAT-5'

• RNA transcript: 5'-UAGCCGUC CUGGAAUUUA-3'

Historical Experiments

• Garrod (1902): Observed hereditary diseases caused by defective enzymes, suggesting that DNA encodes information for enzymes.

• Beadle and Tatum (1941): Demonstrated the "one gene-one enzyme" hypothesis using Neurospora crassa. Mutations in genes led to defects in specific enzymes required for amino acid synthesis.

Central Dogma of Molecular Biology

Flow of Genetic Information

The central dogma describes the flow of genetic information from DNA to RNA to protein.

• Replication: DNA is copied to produce more DNA.

• Transcription: DNA is transcribed into RNA.

• Translation: RNA is translated into protein.

$\text{DNA} \xrightarrow{\text{transcription}} \text{RNA} \xrightarrow{\text{translation}} \text{Protein}$

• DNA is double-stranded; RNA is single-stranded.

• The coding (sense) strand of DNA has the same sequence as the mRNA (except T is replaced by U).

• The template (antisense) strand is used by RNA polymerase to synthesize RNA.

Translation and the Genetic Code

• RNA is translated into a sequence of amino acids using ribosomes and tRNA.

• There are 20 standard amino acids and 4 RNA bases (A, U, G, C).

Codons and the Triplet Code

A codon is a sequence of three nucleotides in mRNA that encodes a specific amino acid.

• With 4 bases and 3 positions, there are $4^3 = 64$ possible codons.

• The code is degenerate: more than one codon can specify the same amino acid.

• Start codon: AUG (codes for methionine)

• Stop codons: UAA, UAG, UGA (signal termination of translation)

First Base	Second Base	Third Base	Amino Acid
U	U	U	Phe (F)
A	U	G	Met (M) - Start
U	A	A	Stop
U	A	G	Stop
U	G	A	Stop

Reading Frames

The reading frame determines how the nucleotide sequence is divided into codons.

• There are three possible reading frames for any mRNA sequence.

• Shifting the reading frame (frameshift mutation) alters the resulting protein.

Example:

• Sequence: 5'-CACGGUCGAUGAGGUUACAUAAC-3'

• Frame 1: His Gly Arg STOP

• Frame 2: Thr Leu His Glu Val Thr STOP

• Frame 3: Met Arg Leu His Thr

Key Experiments in Deciphering the Code

• Crick and Brenner: Demonstrated that the genetic code is read in triplets without spaces; frameshift mutations disrupt the reading frame.

• Nirenberg (1961): Used synthetic mRNA to determine which codons specify which amino acids (e.g., UUU codes for phenylalanine).

Transcription Process in Prokaryotes

The Machinery

• RNA polymerase core enzyme: α and α' (regulatory), β and β' (active site).

• Holoenzyme: Core enzyme plus σ factor (required for initiation).

• RNA polymerase does not require a primer to initiate synthesis.

Initiation

• Promoter regions (-35, -10, +1) upstream of the gene are recognized by the σ factor.

• RNA polymerase binds to the promoter and begins transcription at the +1 site.

Elongation

• Transcription occurs in the transcription bubble.

• σ factor dissociates after initiation; RNA polymerase adds nucleotides to the 3' end of the growing RNA strand.

Termination

• Transcription stops at specific sequences (e.g., hairpin structures formed by inverted repeats followed by a string of U's).

• Hairpin causes RNA polymerase to pause and dissociate from DNA.

Eukaryotic Differences in Transcription

RNA Polymerases

• Eukaryotes have three RNA polymerases (I, II, III), each transcribing different types of genes.

Initiation

• More complex than in prokaryotes; requires multiple transcription factors (TFs) to assemble at the promoter before RNA polymerase can bind.

Compartmentalization

• Transcription occurs in the nucleus; translation occurs in the cytoplasm.

• mRNA is extensively processed before leaving the nucleus.

RNA Processing in Eukaryotes

mRNA Structure

• 5' cap: Addition of a modified guanine nucleotide to the 5' end; protects mRNA from degradation and assists in ribosome binding.

• 3' polyA tail: Addition of 100-200 adenine nucleotides to the 3' end; increases transcript stability.

Primary vs. Mature Transcripts

• Primary transcript (pre-mRNA): Contains both exons (coding regions) and introns (non-coding regions).

• Mature mRNA: Introns are removed by splicing, leaving only exons.

mRNA Splicing

• Spliceosome (snRNPs + proteins) recognizes specific sequences at intron-exon boundaries (GU at 5', AG at 3', and branch point A).

• Spliceosome cuts at the 5' splice site, forms a lariat structure, and joins exons together, excising the intron.

Applications and Examples

Lactase (LCT) Mutation and Persistence

• Lactase persistence is an example of gene regulation and mutation affecting human evolution.

• Mutations in regulatory regions can lead to continued expression of the lactase gene into adulthood.

• Geographic and historical data show the spread of lactase persistence in populations with dairy farming.

Summary Table: Key Differences in Transcription

Feature	Prokaryotes	Eukaryotes
RNA Polymerase	Single type	Three types (I, II, III)
Initiation	σ factor, simple promoter	Multiple TFs, complex promoter
Location	Cytoplasm	Nucleus
RNA Processing	Minimal	5' cap, polyA tail, splicing

Additional info: The notes include historical context and examples (e.g., Garrod, Beadle and Tatum, Crick and Brenner, Nirenberg) to illustrate the development of our understanding of gene expression and the genetic code.