biochem

Steps of Transcription

1. Initiation

   • RNA polymerase binds to a promoter region (specific DNA sequence).

   • DNA unwinds to expose the template strand.

2. Elongation

   • RNA polymerase moves along the DNA template strand.

   • It synthesizes pre-mRNA in the 5' to 3' direction, adding complementary RNA nucleotides (A, U, C, G).

   • Uracil (U) is used instead of thymine (T).

3. Termination

   • RNA polymerase reaches a termination sequence and detaches.

   • The newly formed pre-mRNA is released.

Post-Transcriptional Modifications (Eukaryotes Only)

• 5' Cap: Added to the beginning for stability and ribosome binding.

• Poly-A Tail: Added to the 3’ end to protect mRNA from degradation.

• Splicing: Introns (non-coding regions) are removed; exons (coding sequences) are joined.

Translation (RNA to Protein)

Overview

• Translation is the process of converting mRNA into a polypeptide chain (protein).

• Occurs in the cytoplasm on ribosomes.

Key Components

• mRNA: Carries codons (3-nucleotide sequences).

• tRNA (transfer RNA): Brings amino acids to the ribosome; has anticodons that pair with mRNA codons.

• Ribosome: Made of rRNA and proteins; has A (aminoacyl), P (peptidyl), and E (exit) sites.

Steps of Translation

1. Initiation

   • Small ribosomal subunit binds to mRNA.

   • Start codon (AUG) is recognized.

   • First tRNA carrying methionine binds to AUG.

   • Large ribosomal subunit attaches to form a functional ribosome.

2. Elongation

   • New tRNA enters A site matching next codon.

   • Peptide bond forms between amino acids.

   • Ribosome shifts, tRNA moves from A → P → E site.

   • tRNA in E site exits the ribosome.

3. Termination

   • Stop codon (UAA, UAG, UGA) is reached.

   • Release factors bind; the polypeptide is released.

   • Ribosome disassembles.

Genetic Code

• Redundant but not ambiguous (multiple codons per amino acid, but each codon codes for only one amino acid).

• Universal across almost all organisms.

Initiation

• Begins at the origin of replication (Ori).

• Initiator proteins recognize and bind to the origin, recruiting other enzymes.

• Helicase unwinds the DNA helix by breaking hydrogen bonds between bases.

• Single-strand binding proteins (SSBs) bind to and stabilize the unwound DNA.

• Topoisomerase (or gyrase in prokaryotes) relieves supercoiling ahead of the replication fork.

Elongation

• Primase synthesizes a short RNA primer, providing a free 3’ OH group for DNA polymerase.

• DNA Polymerase III adds nucleotides to the 3’ end of the primer in the 5’→3’ direction.

• On the leading strand, synthesis is continuous toward the replication fork.

• On the lagging strand, synthesis is discontinuous, forming Okazaki fragments away from the fork.

• DNA Polymerase I removes RNA primers (5’→3’ exonuclease activity) and fills in DNA.

• DNA Ligase seals the nicks between Okazaki fragments to form a continuous strand.

Proofreading and Fidelity

• DNA Polymerase III has 3’→5’ exonuclease activity, allowing it to remove incorrect nucleotides.

• Mismatch repair systems correct any errors missed by polymerases.

• Overall error rate after proofreading and repair is approximately 1 in 10⁹ nucleotides.

Class 35: Gene Regulation – Part 1

Why Gene Regulation is Important

• Allows cells to respond to environmental changes and conserve energy.

• Ensures genes are expressed at the right time, place, and level.

• Enables cell specialization in multicellular organisms through differential gene expression.

Levels of Gene Regulation

• Transcriptional regulation: Controls when and how often a gene is transcribed (most common).

• Post-transcriptional regulation: Involves mRNA splicing, editing, transport, and degradation.

• Translational regulation: Controls the efficiency and rate of mRNA translation into protein.

• Post-translational regulation: Modifies proteins after translation (e.g., phosphorylation, degradation).

Prokaryotic Gene Regulation

The Operon Model

• An operon is a cluster of functionally related genes controlled by a single promoter and regulated together.

• Consists of:

  • A promoter, where RNA polymerase binds.

  • An operator, where regulatory proteins (repressors or activators) bind.

  • Structural genes encoding proteins with related functions.

Lac Operon (Inducible System)

• Involved in lactose metabolism in E. coli.

• Normally off; turned on in the presence of lactose.

• LacI gene encodes a repressor that binds the operator and blocks transcription.

• Allolactose (a derivative of lactose) binds the repressor, causing it to release from the operator, allowing transcription.

• Catabolite repression:

  • When glucose is low, cAMP levels rise.

  • cAMP binds CAP (catabolite activator protein), forming a complex that enhances RNA polymerase binding to the promoter.

  • This ensures that the cell prefers glucose over lactose when both are present.

Trp Operon (Repressible System)

• Involved in tryptophan synthesis.

• Normally on; turned off when tryptophan is abundant.

• Tryptophan acts as a corepressor, binding the trp repressor and allowing it to attach to the operator to block transcription.

• Also regulated by attenuation: formation of stem-loop structures in mRNA depending on tryptophan levels controls whether transcription continues.

Class 36: Gene Regulation – Part 2

Eukaryotic Gene Regulation Overview

• More complex due to chromatin structure, multicellularity, and compartmentalization.

• Regulation occurs at multiple stages, from chromatin remodeling to post-translational modifications.

Chromatin Structure and Epigenetics

• Euchromatin is loosely packed and transcriptionally active.

• Heterochromatin is densely packed and transcriptionally inactive.

• Histone modification affects DNA accessibility:

  • Acetylation (via HATs) loosens chromatin by neutralizing positive charges on histones.

  • Deacetylation (via HDACs) tightens chromatin and represses transcription.

  • Methylation of DNA (usually on CpG islands) can silence gene expression.

• Epigenetic regulation refers to heritable changes in gene expression not caused by changes in DNA sequence.

Transcriptional Regulation in Eukaryotes

• General transcription factors help RNA polymerase bind to core promoters (e.g., TATA box).

• Specific transcription factors bind enhancers (activators) or silencers (repressors) to regulate gene expression in a cell-type or signal-specific manner.

• Mediator complex bridges transcription factors at enhancers/silencers and RNA polymerase at the promoter.

• DNA looping allows enhancers to influence promoters from far away.

Post-Transcriptional Regulation

• Alternative splicing allows a single gene to produce multiple protein isoforms by including/excluding specific exons.

• RNA editing can change nucleotide sequences after transcription (e.g., A→I editing).

• mRNA transport and localization can control when and where translation occurs.

• mRNA stability determines how long mRNA is available for translation; influenced by 5’ cap, 3’ poly-A tail, and binding proteins.

• RNA interference (RNAi):

  • siRNAs and miRNAs are small non-coding RNAs that bind to complementary mRNA sequences.

  • Binding results in mRNA degradation or inhibition of translation, depending on the degree of complementarity.

Translational and Post-Translational Regulation

• Translation can be regulated by:

  • Phosphorylation of initiation factors.

  • Regulatory proteins that bind the 5’ UTR or 3’ UTR of mRNA.

• Post-translational modifications include:

  • Phosphorylation, which can activate or deactivate proteins.

  • Ubiquitination, which marks proteins for degradation by the proteasome.

  • Glycosylation, methylation, and other modifications affecting localization, stability, or function.

Comparison of Prokaryotic vs. Eukaryotic Gene Regulation

• Prokaryotes often regulate gene clusters (operons) at the transcriptional level.

• Eukaryotes regulate single genes, often involving enhancers, chromatin remodeling, and alternative splicing.

• Eukaryotic genes are separated from ribosomes (transcription and translation are uncoupled), enabling more regulation layers.