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.