Fundamentals of Biochemistry — Chapter 26: Transcription and RNA Processing
Central Dogma of Molecular Biology
Key Processes:
- Replication: The process of duplicating DNA.
- Transcription: The synthesis of RNA from a DNA template.
- Translation: The conversion of RNA sequences into protein.
Key Molecules:
- RNA: Nucleic acid involved in various biological roles.
- Protein: Large biomolecules made up of amino acids, essential for cellular structure and function.
- DNA: Deoxyribonucleic acid, the molecule that carries genetic information.
Major Types of RNA in Cells
Cells contain three major types of RNA:
- Messenger RNA (mRNA):
- Size: Varies (hundreds to thousands of nucleotides).
- Function: Directs protein synthesis by carrying the genetic code from DNA to ribosomes.
- Ribosomal RNA (rRNA):
- Size: 120-4718 nucleotides.
- Function: Constitutes two-thirds of ribosome mass; essential for ribosome structure and catalytic activity in translation.
- Transfer RNA (tRNA):
- Size: 54-100 nucleotides.
- Function: Delivers amino acids to ribosomes during translation.
Other Types of Noncoding RNA
Small interfering RNA (siRNA):
- Size: 20-25 nucleotides.
- Function: Sequence-specific inactivation of mRNA, playing roles in gene regulation and silencing.
Micro RNA (miRNA):
- Size: 20-25 nucleotides.
- Function: Also involved in sequence-specific inactivation of mRNA, impacting gene expression.
Large intergenic noncoding RNA (lincRNA):
- Size: Up to 17,200 nucleotides.
- Function: Plays a role in transcriptional control.
Small nuclear RNA (snRNA):
- Size: 60-300 nucleotides.
- Function: Involved in RNA splicing.
Small nucleolar RNA (snoRNA):
- Size: 70-100 nucleotides.
- Function: Involved in the sequence-specific methylation of rRNA.
Mechanisms of Transcription
Overview of RNA Polymerase Reactions
RNA Polymerase (RNAP) Mechanism:
- Utilizes a DNA template for RNA synthesis.
- Direction of Synthesis: Synthesizes RNA in the 5' to 3' direction and uses the 3’ –OH as a nucleophile to attack the α-phosphate group of incoming NTPs (nucleotide triphosphates).
DNA Polymerase:
- Similar mechanism in DNA synthesis with template strand.
- Primer Requirement: DNA polymerase requires a primer; RNAP does not.
Polymerase Active Sites
Mg2+ Ions:
- Both DNA and RNA polymerases have two magnesium ions (Mg²⁺) in the active site, coordinated by two conserved aspartate residues.
Prokaryotic Transcription Overview
Prokaryotes possess a single RNA polymerase enzyme responsible for synthesizing all types of RNA except primers for DNA replication.
Sense and Antisense Strands
Sense (Coding) Strand:
- The RNA sequence corresponds to the DNA coding strand, with the substitution of uracil (U) for thymine (T).
Antisense (Noncoding) Strand:
- The RNA sequence is complementary to the antisense strand, also substituting U for T.
Initiation: Binding of the RNAP holoenzyme to DNA at promoter regions involves core subunits.
Key Features of the Initiation Process
Holoenzyme vs Core Enzyme:
- The holoenzyme is more specific to the promoter sequence, while the core enzyme has lower affinity for dsDNA.
Promoter Search:
- The holoenzyme slides along dsDNA until it encounters a promoter region, initiating transcription.
Sigma Factors:
- Different sigma factors regulate the expression of specific genes by binding to unique promoter sequences.
Operon Structure and Selected Promoters
Promoter Elements:
- -35 Region: Consensus sequence around TTGACA.
- -10 Region (Pribnow Box): Consensus sequence around TATAAT.
- Initiation Site (+1): Site where transcription begins.
Properties of RNA Polymerase
Processivity: RNA polymerase is highly processive.
Transcription Speed: Approximately 20-50 nucleotides per second, slower than DNA polymerase's 1000 nucleotides per second.
Fidelity of Transcription: Lower fidelity compared to replication, with about 1 error in 10,000 nucleotides, while DNA replication has an error rate of 1 in 10^8 to 10^10.
Transcription Bubble Dynamics
Formation: Characterized by unwinding of the DNA helix to expose the template strand.
Structure: Involves the formation of over- and under-winding regions adjacent to the transcription machinery.
Transcription Termination Mechanisms
Intrinsic Terminator:
- Consists of a G + C-rich region followed by a palindrome, leading to the formation of a stable stem-loop structure in RNA, which causes separation of RNA from the DNA template.
Rho Factor-dependent Termination:
- Rho acts as a helicase that unwinds RNA-DNA complexes by translocating along RNA, requiring recognition sequences and rut sites on the RNA.
- Mechanism:
1. Rho hexamer binds to the C-rich sequence (primary binding site).
2. Transition from open-ring to closed-ring state, becoming catalytically active.
3. Translocates along the RNA to exert mechanical force, disrupting the transcription elongation complex (TEC).
Summary of Prokaryotic RNA Transcription
Resembles DNA polymerase in structure and mechanism.
Initiation involves binding of RNA polymerase to DNA promoters.
Transcription can occur simultaneously by multiple polymerases.
Termination depends on specific sequences in the DNA or the activity of the Rho factor.