Comprehensive Study Notes: Gene Expression, Transcription, and Translation

Nature and Function of the Gene 2: Gene Expression and Regulation

  • The central dogma of molecular biology defines the flow of genetic information: DNA→RNA→Protein\text{DNA} \rightarrow \text{RNA} \rightarrow \text{Protein}.
  • Gene expression involves several key stages:     - Transcription.     - mRNA processing (distinctive in eukaryotes).     - Control of expression.     - Translation.
  • Eukaryotes vs. Prokaryotes:     - In eukaryotes, transcription occurs in the nucleus. Pre-mRNAs undergo processing into mature mRNAs before being exported to the cytoplasm for translation.     - In prokaryotes, mRNA processing is absent. Transcription and translation occur simultaneously within a single compartment.

Prokaryotic Transcription and RNA Polymerase

  • Transcription is the process of converting DNA to mRNA via DNA-dependent RNA polymerases (RNA polymerases).
  • RNA polymerases incorporate nucleotides into a new strand of RNA using a DNA template.
  • Bacterial RNA Polymerase Structure: Consists of six subunits:     - Two α\alpha subunits (colored green in diagrams).     - One β\beta subunit (colored blue).     - One β′\beta' subunit (colored pink).     - One ω\omega subunit (colored yellow).     - One σ\sigma (sigma) subunit: This subunit is relatively weakly bound and can dissociate from the core enzyme.

Prokaryotic Promoter Sequences and Initiation

  • Transcription begins at a "promoter" located upstream of the RNA-coding sequence and halts at a "terminator" located downstream.
  • Promoter Conservation: Prokaryotic promoter sequences are highly conserved. They contain two primary conserved regions (colored red in sequence alignments):     - −35-35 element: A conserved sequence region.     - −10-10 element: Also known as the "Pribnow box."     - These elements are typically separated by a specific distance (e.g., < 16-18\,bp or < 20\,bp).
  • Promoter Examples from T. maritima and E. coli:     - TM0373: TTACAA\text{TTACAA}… 18 bp18\,bp… TATAAT\text{TATAAT}     - TM1016: TTAAAA\text{TTAAAA}… 16 bp16\,bp… TTTAAT\text{TTTAAT}     - TM1272/TM1429: CTTGACA\text{CTTGACA}… 17 bp17\,bp… TTTAAT/TATAAT\text{TTTAAT/TATAAT}     - Consensus sequence for E. coli: TTGACA\text{TTGACA}… 17 bp17\,bp… TATAAT\text{TATAAT}.
  • Initiation Process:     - RNA polymerase initially binds non-specifically to DNA.     - It migrates along the molecule until the σ\sigma (sigma) subunit interacts specifically with the −35-35 and −10-10 promoter elements.     - The polymerase unwinds the DNA at the initiation site, and transcription begins.
  • Elongation:     - Following initiation, the σ\sigma subunit dissociates from the core polymerase.     - The core polymerase migrates along the DNA and elongates the growing RNA chain until it reaching a termination point.

Transcription Termination in Prokaryotes

  • Stem-loop Termination: Signaled by a G-C\text{G-C} rich inverted repeat followed by a sequence of 77 A residues.     - This results in the formation of a stable "stem-loop" mRNA structure.     - This structure causes the mRNA to dissociate from the DNA template, terminating transcription.
  • Rho-dependent Termination: Alternatively, some prokaryotic genes require a specific "termination protein" called Rho to signal the end of transcription.

Eukaryotic Transcription and RNA Polymerase II

  • Eukaryotes possess multiple RNA polymerases (I, II, and III).
  • RNA Polymerase II (Pol II): Responsible for transcribing protein-coding genes.     - Yeast RNA Polymerase II consists of 12−1712-17 subunits (9 of which are conserved with the bacterial polymerase).     - Two specific subunits (β\beta-like) are similar to bacterial polymerases.
  • RNA Pol I and III: Share similarities to Pol II but utilize different mechanisms and transcription factors.

The Eukaryotic Preinitiation Complex and Initiation

  • Transcription initiation begins with the formation of a preinitiation complex (PIC) involving multiple transcription factors (TFs) and protein-DNA interactions.
  • Steps of Assembly:     - TFIID: Binds to promoter consensus sequences like the TATA box (TATAATATA A) or BRE. It recognizes these sites via the TBP (TATA-binding protein) and TAFs (transcription-associated factors). The TATA box is typically located at −25-25.     - Other Factors: RNA polymerase and additional transcription factors (TFIIB, TFIIE, TFIIF, and TFIIH) bind to form the pre-initiation complex.     - Mediator Protein: A large protein complex that binds to the PIC to regulate activity.
  • Transition to Elongation:     - The C-terminal domain (CTD) of RNA polymerase II undergoes phosphorylation.     - This phosphorylation releases the Mediator and general transcription factors, allowing Polymerase II to catalyze RNA synthesis in association with elongation and processing factors.

Post-Transcriptional mRNA Processing (Eukaryotic)

Processing occurs in three main stages to convert pre-mRNA into mature mRNA:

1. 5' Capping
  • A 5′5' cap is formed by adding a GTP in a reverse orientation to the 5′5' end of the mRNA.
  • This creates a unique 5′5'-to-5′5' linkage (different from the standard 5′5'-to-3′3' phosphodiester linkage).
  • The added Guanine (G) is methylated at the N-7 position, forming 7-Methylguanosine.
  • Methyl groups may also be added to the riboses of the first one or two nucleotides of the original mRNA.
  • Functions: Stabilizes mRNA and plays a crucial role in translation initiation.
2. 3' Polyadenylation (Addition of Poly-A Tail)
  • Signals: The process is triggered by polyadenylation signals: an upstream AAUAAA sequence and downstream elements (often G-U rich or U-rich) surrounding a CA cleavage site.
  • Cleavage: An endonuclease cleaves the pre-mRNA 1010 to 3030 nucleotides downstream of the AAUAAA sequence, usually at the CA site.
  • Poly-A Polymerase: This enzyme adds a poly-A tail consisting of approximately 200200 Adenine (A) residues to the newly created 3′3' end.
  • Functions: Stabilizes mRNA and assists in translation regulation.
3. Intron Splicing
  • Introns: Non-coding sequences within the pre-mRNA that must be removed.
  • Exons: Sequences that are joined together to form the mature mRNA.
  • Mechanism:     - Cleavage at the 5′5' splice site (SS).     - The 5′5' end of the intron is joined to an internal Adenine within the intron, called the branch point.     - Cleavage occurs at the 3′3' splice site with simultaneous ligation of the exons.     - The excised intron is released.
  • Spliceosome: A complex responsible for splicing, composed of snRNPs (small nuclear ribonuclear particles).     - snRNPs consist of snRNAs (U1, U2, U4, U5, U6) and 6−106-10 protein molecules.
  • Ribozymes: Catalytic RNA species that drive the splicing process.

Components of General Translation

  • Translation: The process of converting mRNA sequence into a polypeptide chain (protein), facilitated by ribosomes.
  • mRNAs and Polysomes: In eukaryotes, mRNAs are often translated by a series of multiple ribosomes known as "polysomes."
  • tRNA (Transfer RNA) Structure:     - Approximately 70−8070-80 nucleotides long.     - Features a cloverleaf structure due to complementary base pairing (loops).     - Contains modified bases such as pseudouridine and dihydrouridine.
  • Aminoacyl tRNA Synthetases:     - A family of enzymes (approximately 4040 species per cell) that catalyze the linkage of a specific amino acid to its corresponding tRNA.     - Process:         1. An amino acid (e.g., alanine) is coupled with AMP (forming aminoacyl AMP).         2. The aminoacyl AMP combines with the tRNA to form aminoacyl tRNA.

Ribosome Structure and Function

  • The ribosome is a molecular machine that interprets the mRNA code.
  • Abundance:     - E. coli: approximately 20,00020,000 per cell (constituting 25%25\% of dry weight).     - Dividing mammalian cell: approximately 1×1071 \times 10^7 per cell.
  • Subunit Composition:     - Prokaryotic (70S) Ribosome: Consists of a 50S large subunit (23S23S and 5S5S rRNAs + 3434 proteins) and a 30S small subunit (16S16S rRNA + 2121 proteins).     - Eukaryotic (80S) Ribosome: Consists of a 60S large subunit (28S28S, 5.8S5.8S, and 5S5S rRNAs + ~4646 proteins) and a 40S small subunit (18S18S rRNA + 3333 proteins).
  • Functional Sites: Every ribosome has three binding sites for tRNA:     - A site (Aminoacyl).     - P site (Peptidyl).     - E site (Exit).
  • rRNA Roles: rRNAs provide the structural scaffold for proteins and possess catalytic activity (e.g., peptidyl transferase activity in the large subunit).

Stages of Translation

  1. Initiation: The ribosome binds to the mRNA at the start codon (usually AUG\text{AUG}, which codes for fMet in prokaryotes).
  2. Elongation: The polypeptide chain grows as the ribosome moves along the mRNA, adding amino acids one by one based on the codon sequence.
  3. Termination: When the ribosome encounters a stop codon, the completed polypeptide is released and the ribosome subunits dissociate.

Recommended Academic Resources

  • Key Textbooks:     - Cooper, G.M. & Hausman, R.E. (2019). The Cell: A Molecular Approach (8th ed.).     - Lodish et al. (2012). Molecular Cell Biology (7th ed.).     - Alberts et al. (2015). Molecular Biology of the Cell (6th ed.).
  • Online Resources:     - DNA Learning Centre (Cold Spring Harbor): https://dnalc.cshl.edu/websites/     - DNA from the Beginning: http://www.dnaftb.org/     - Bozeman Science YouTube: Transcription and Translation tutorials.