DNA-directed RNA Synthesis and Processing

DNA-directed RNA Synthesis

Overview of Transcription

  • Transcription: The process of synthesizing RNA from a DNA template.

  • Key Concepts:

    • mRNA transcript generation

    • Directionality of synthesis is from 5' to 3'

    • Involves the enzyme RNA polymerase

Steps in Transcription (in all organisms)

  1. Promoter Recognition:

    • RNA polymerase identifies promoter regions on the DNA.

  2. DNA Unwinding:

    • The double-stranded DNA unwinds to expose the coding regions.

  3. RNA Synthesis:

    • RNA polymerase synthesizes RNA by adding nucleotides to the growing RNA strand.

    • Direction of RNA synthesis: 5' to 3'.

  4. Termination:

    • RNA polymerase stops synthesis at specific termination signals.

    • The new RNA transcript is released, and RNA polymerase detaches from the DNA.

Structure and Function of RNA Polymerase

Prokaryotic RNA Polymerase

  • Holoenzyme Composition:

    • Core Enzyme:

    • α (alpha) subunits (2 copies): Assist in enzyme assembly and interaction with regulatory proteins.

    • β (beta): Catalytic center that binds ribonucleotides.

    • β′ (beta prime): Binds the DNA template.

    • ω (omega): Stabilizes the enzyme.

    • σ (sigma) Factor:

    • Directs the core enzyme to specific promoters ensuring correct transcription initiation.

Eukaryotic RNA Polymerases

  • Three Main Types:

    1. RNA Polymerase I: Synthesizes most rRNA.

    2. RNA Polymerase II: Synthesizes mRNA and some small nuclear RNAs (snRNA).

    3. RNA Polymerase III: Synthesizes tRNA, 5S rRNA, and other small RNAs.

  • Subunit Composition:

    • Each polymerase has approximately 12–17 protein subunits.

    • Contains two large catalytic subunits analogous to beta and beta prime in prokaryotes.

    • Includes several smaller subunits shared among different polymerases and additional accessory factors (transcription factors) required for promoter recognition and regulation.

Stages of Transcription

Initiation

  • Involves promoter binding and RNA polymerase assembly.

  • Influenced by cis-acting and trans-acting regulatory elements.

Elongation

  • Addition of RNA nucleotides to the 3' end of the growing RNA transcript, accounting for the polarities of both the template and synthesis.

Termination

  • Mechanisms:

    • Formation of stem-loop (hairpin) structures during transcription in prokaryotes.

    • Interaction with terminator proteins.

Transcription in Eukaryotes

  • Occurs in the nucleus and involves the coordination of various processes:

    • Transcription

    • 5'-end capping

    • 3'-end modifications

  • RNA Process:

    • RNA transcripts are processed before being exported from the nucleus to the cytoplasm.

Strands of DNA During Transcription

  • Sense Strand (Coding Strand):

    • Also known as the coding strand.

    • Contains the same sequence as the RNA transcript (thymine instead of uracil).

    • Located upstream of the gene's coding region.

  • Antisense Strand (Template Strand):

    • Also known as the template strand.

    • RNA polymerase travels along this strand from 3' to 5' directing RNA synthesis in the 5' to 3' direction.

Major Differences in Transcription: Prokaryotes vs Eukaryotes

Aspect

Eukaryotes

Prokaryotes

Regulation

Involves many cis-acting DNA and trans-acting protein factors

Operons are common

Initiation

Requires DNA/chromatin remodeling, occurs in the nucleus

Occurs in cytoplasm, transcription & translation are coupled

DNA Remodeling

Involves nucleosome uncoiling

Relaxed supercoiling

Complexity

More complex with enhanced interactions

Generally simpler

Post-Transcriptional Modifications (mRNA Processing)

Basic Overview

  • Occurs in the nucleus and includes multiple modifications to pre-mRNA:

    • 5'-end capping

    • Splicing

    • 3'-end cleavage and polyadenylation (adding a poly-A tail)

5’-end Capping

  • 7-methylguanosine (7-mG) Cap:

    • Added to the 5'-end of pre-RNA early in the transcription process after approximately 20 nucleotides have been synthesized.

    • Protects mRNA from degradation by nucleases.

    • Facilitates ribosome binding during translation.

3’-end Polyadenylation

  • Poly-A Tail:

    • Added to 3' end of the mRNA by poly-A polymerase, which adds up to 250 adenylic acid residues.

    • Stabilizes the mRNA and marks it for export to the cytoplasm.

Splicing

  • Involves removal of introns and ligation of exons to form the mature mRNA.

  • Components:

    • Intronic micro-RNAs and transposons may be involved.

    • Formation of Open Reading Frames (ORFs) from joined exons.

    • Alternative Splicing: Allows the production of different proteins from the same gene by including or excluding certain exons during the splicing process.

Control of Transcription

  • Key Components:

    • Enhancers and silencers (cis-regulatory elements) bind transcription factors and influence transcription levels.

    • Induction and repression mechanisms regulate gene expression.

Translation Overview

  • Purpose: mRNA-directed synthesis of proteins.

Steps of Translation

  1. Initiation:

    • Prokaryotes: Shine-Dalgarno sequence recognition and binding by the ribosome.

    • Eukaryotes: Cap recognition and Kozak sequence.

  2. Elongation:

    • Determined by the codons in mRNA and the genetic code.

    • Involves tRNA anticodon recognition of codons, followed by amino acid polymerization through peptide bond formation.

  3. Termination:

    • Stop codon recognition signals the end of translation, involving termination factors without corresponding tRNA.

The Genetic Code

  • The sequence of nucleotides in mRNA is translated into amino acids with the help of ribosomes and transfer RNA (tRNA) molecules.

Codons

  • Definition: A codon consists of three sequential ribonucleotide letters that encode a single amino acid.

  • Total Number of Codons: 64

    • Sense Codons: 61 sense codons encoding 20 amino acids; one also functions as a start signal.

    • Nonsense Codons: 3 codons (UAG, UAA, UGA) that do not encode an amino acid and function as stop signals.

Open Reading Frame (ORF)

  • Defined as a segment of RNA starting with a start codon and ending just before a stop codon, not including any stop codons.

    • Initiator Codon: Usually AUG which encodes Methionine in eukaryotes and N-formylmethionine (fmet) in bacteria.

    • Termination Codons: UAG, UAA, UGA, indicating the end of translation and not recognized by tRNA.

Properties of the Genetic Code

  • Unambiguous: Every sense codon specifies only one amino acid.

  • Degenerate: A single amino acid can be specified by more than one codon.

  • Universal: Common across nearly all organisms.