RNA Translation

E. coli Transcription Unit

  • Components of Transcription Unit:

    • Start: -35 Region, -10 Region, +1

    • Promoter and Terminator sequences essential for proper initiation and termination

  • Transcription Elements:

    • RNA synthesis starts at +1 position (PPP5 AUG UAA)

    • Polypeptide chain formatted: H2N (N-terminus) to COOH (C-terminus)

Transcriptional Termination

  • Types of Termination:

    • Intrinsic (p-independent) vs. Rho-dependent (p-dependent)

  • Key features: (a) intrinsic termination involves RNA hairpin formation, leading to transcription termination once the RNA polymerase encounters a terminator sequence. (b) Rho-dependent termination involves the Rho helicase binding to a rut site in RNA, assisting in disengagement of RNA from the RNA polymerase.

  • Diagrammatic representation indicates crucial sequences and processes.

Transcription in Prokaryotes vs. Eukaryotes

  • Prokaryotes:

    • Transcription occurs in cytoplasm; coupled with translation.

  • Eukaryotes:

    • Transcription occurs in nucleus, and translation occurs in cytoplasm; they are uncoupled.

    • Eukaryotic mRNAs undergo processing post-transcription.

Eukaryotic Promoter Recognition

  • Promoters recognized by RNA Polymerase II:

    • Key sequences: -30 (TATA box) and +1

    • Regulatory sequences influence transcription initiation.

Initiation of Transcription by RNA Pol II

  • Core promoter elements recruit transcription factors:

    • TATA-box recognized by TBP, facilitating assembly of transcription factors and RNA Pol II.

  • Process of initiation:

    1. DNA unwinding occurs, forming a pre-initiation complex to an open initiation complex.

    2. Transcription of first few nucleotides takes place, followed by phosphorylation of the polymerase CTD, allowing entry into elongation phase.

Inhibitors of Transcription

  • Key inhibitors:

    • Rifampicin: Inhibits initiation of RNA synthesis.

    • Actinomycin D: Inhibits elongation process.

  • Eukaryotes possess three RNA polymerases, sensitized by α-Amanitin.

RNA Processing in Eukaryotes

  • Transcription processed in nucleus before exporting:

    • Steps:

      1. 5’ capping

      2. Splicing

      3. Poly(A) tail addition

  • Prokaryotes do not undergo these processes for mRNA.

Capping of Eukaryotic mRNAs

  • 5' cap structure consists of an N7-methyl G cap linked through a unique triphosphate linkage.

  • Role of 5' cap:

    • Essential for gene expression, protection from exonucleases, and includes methylation of first two nucleotides at 2’OH position.

Splicing in Eukaryotic mRNAs

  • Splicing removes introns, sequences absent in mature mRNA.

  • Some introns possess self-splicing capabilities.

Spliceosome Functionality

  • Spliceosome, a nuclear complex, contains snRNPs (small nuclear ribonucleoproteins) made from RNA and proteins.

  • RNA component's importance for catalysis in splicing.

Poly A Tail Addition

  • All mRNAs feature a 3' A-residue stretch not encoded in DNA.

  • Cleavage and poly(A) machinery recognizes AAUAAA sequence to enhance gene expression and protect mRNA from degradation.

Overview of Eukaryotic mRNA Processing

  • Example: Ovalbumin gene

  • Representation of transcription and processing steps emphasizing intron removal.

Introduction to Translation

  • Genetic information from DNA transcribed into mRNA; requires conversion to amino acids (protein).

  • Ribosomal machinery facilitates translation, charged tRNA acts as connector.

The Adapter Hypothesis

  • Francis Crick proposed the concept of an adapter molecule for protein synthesis.

Charged tRNA Structure

  • tRNA adopts cloverleaf secondary structure and elbow-shaped tertiary structure; contains anticodon for mRNA pairing.

Ribosome Composition

  • Ribosome consists of two subunits—comprised of RNA and protein; larger in eukaryotes.

  • Key operational units in cytoplasm for protein synthesis.

Charged tRNA Functionality

  • Charged tRNA links mRNA to amino acids through anticodon-codon complementarity.

  • Charging process performed by aminoacyl-tRNA synthetases and requires ATP.

Direction of Translation

  • Translation proceeds from N-terminus to C-terminus; mRNA is read in a 5' to 3' direction.

Codon Features

  • Codon characteristics: triplet, non-overlapping, no punctuation, degenerate, and universal nature.

  • Minimum nucleotides required to code for 20 amino acids is 3.

The Genetic Code

  • 61 sense codons corresponding to 20 amino acids and 3 stop codons.

  • Example codon table correlating nucleotides with specific amino acids.

Codon Analysis

  • Provided sequence for codon translation, identifying the peptide structure with start codon highlighted.

Translation Steps

  • Translation involves three phases: initiation, elongation (decoding, peptide bond formation, translocation), and termination.

Initiation Requirements

  • Translation initiation requires ribosomal subunits, mRNA, initiation factors, and first charged tRNA fMet-tRNA.

Differences in Initial tRNA

  • Prokaryotes initiate translation with N-formyl methionine tRNA; eukaryotes utilize standard methionine.

Elongation Cycle Steps

  • During elongation (steps E, P, A) the ribosome engages in multiple processes including decoding and peptide bond formation.

Factors in Elongation

  • Requires a 70S ribosome and elongation factors with GTP for accurate protein synthesis during elongation phases.

Peptide Bond Formation in Elongation

  • Peptide bond formation is spontaneous, ribosome catalyzes this without necessary additional factors.

Translocation in Elongation

  • Movement of tRNAs through ribosomal sites requires elongation factors and GTP.

Termination Phase

  • Termination requires a 70S ribosome, release factors which add water for protein release and dissociation of ribosomal subunits.

GTP Usage in Translation Steps

  • Evaluation of GTP utilization in translation steps for correct understanding and implications.

Co-transcriptional Translation in Prokaryotes

  • Prokaryotic translation occurs concurrently with transcription, allowing ribosomes to attach and translate mRNA rapidly.

Post-translational Modifications (PTM)

  • Overview of PTMs:

    • Include proteolytic cleavage, amino acid modifications, carbohydrate attachments, prosthetic group additions impacting protein functionality.

Protein Targeting to ER

  • Recognition of signal sequence by SRP for targeting proteins to the ER, with co-translational insertion process.

Ubiquitin-Targeted Proteins

  • Ubiquitin chains modify proteins for degradation by the proteasome:

    • Activation by E1 enzyme using ATP, transfer to E2, targeted by E3 ligase to a lysine residue for tagging.

Proteasomal Degradation

  • 26S proteasome's structure and function in degrading poly-ubiquitinated proteins for cellular regulation.

Regulation of Gene Expression

  • Explanation of cellular differentiation despite identical DNA; constitutive and inducible genes.

  • Overview of gene expression regulation's significance in molecular biology.

Point of Gene Regulation

  • Gene regulation can occur at multiple stages leading to mature proteins; primarily at transcription initiation in prokaryotes.

Core Promoter Variations

  • Gene regulation often influenced by variations in core promoter sequences, affecting sigma factor binding in operons.

Activation and Repression of Gene Expression

  • Regulation can be either positive (activators) or negative (repressors).

Study of the Lac Operon

  • Importance of regulating lactose metabolism in E. coli to optimize enzyme use based on nutrient availability.

Structure of Prokaryotic Operons

  • Operons feature closely linked functional genes, promoters, and regulatory sites crucial for transcriptional control.

Lac Repressor Mechanism

  • Lac repressor structure and function in inhibiting lac operon transcription in absence of lactose.

Inducers of Lac Operon

  • Role of lactose and allolactose as an inducer, including alternative synthetic inducers like IPTG.