Transcription_Prok_SP25_ebae94b4e86abb0ca679833c96f35553

Genotype and Phenotype

The genotype of an organism specifies its phenotype. This involves the genetic makeup (genotype) determining physical traits (phenotype). The relationship between genotype and phenotype is fundamental in genetics, as the genotype carries the information that translates into the observable characteristics of an organism, influenced by environmental factors.

Essential Characteristics of Hereditary Molecules

• Replication & Transmission: Hereditary molecules, such as DNA, must be capable of replicating faithfully and being transmitted to the next generation to ensure continuity of genetic information. This requires precise mechanisms to prevent mutations.

• Information Storage: They must store genetic information, which includes codes for proteins and regulatory sequences that dictate when and how genes are expressed, essential for the development and function of an organism.

• Genetic Variation: Variation in genetic information leads to differences among organisms, which can affect traits such as appearance, behavior, and susceptibility to diseases. This variation is crucial for evolution and adaptation.

• Phenotypic Expression: The expression of genes results in observable traits, which can be influenced by interactions between multiple genes (polygenic inheritance) and environmental factors (phenotypic plasticity).

RNA Transcription in Prokaryotes

Transcription Overview

RNA transcription involves the process where messenger RNA (mRNA) is synthesized from a DNA template. This process is vital for translating genetic information into functional proteins.

• Electron Micrograph: Visual evidence showing RNA being transcribed at a microscopic level provides insight into the dynamic nature of gene expression.

Learning Objectives

After the lecture, you should be able to:

• Define key terminology related to RNA synthesis.

• List the essential requirements for RNA synthesis, acknowledging the significance of promoters, RNA polymerase, and transcription factors.

• Describe the roles of sigma factors and promoters in the initiation of transcription, including how they contribute to the specificity of gene expression.

• Explain the structure and function of E. coli RNA polymerase and how it assembles with other factors to form the holoenzyme necessary for initiating transcription.

• Understand the mechanisms of transcription initiation, elongation, and termination, and the significance of each step in gene expression.

Transcription Definition

The process of synthesizing ribonucleic acid (RNA) from a DNA template, catalyzed by RNA polymerase. This process engenders the production of mRNA, which carries genetic information to the ribosomes for protein synthesis.

• Historical Context: Initial transcription studies were conducted in E. coli, where mRNA was first discovered, highlighting the evolutionary importance of understanding transcription in model organisms.

• RNA Structure: RNA consists of ribonucleotides that form a single strand, differing from DNA in that it contains uracil instead of thymine and has a hydroxyl group on the ribose sugar. This structure directly influences its functionality during transcription.

Requirements for RNA Synthesis

• A gene (DNA template) with a promoter

• RNA polymerase

• Transcription factors: Proteins that assist in guiding RNA polymerase to the promoter and facilitating the initiation of transcription.

• Nucleoside triphosphates (NTPs): The building blocks that RNA polymerase incorporates into the growing RNA strand.

• Visual Aid: Electron micrograph highlighting transcription of ribosomal RNA genes to demonstrate transcribing mechanisms.

Structure of E. coli RNA Polymerase

• Core Enzyme Role: Responsible for synthesizing RNA, polymerizing ribonucleoside triphosphates in a 5’ to 3’ direction, enabling rapid and efficient RNA synthesis without the need for a primer.

• Lack of Primer: RNA polymerase does not require a primer to initiate transcription, unlike DNA polymerases.

• Inactive Form: Cannot begin transcription alone and requires additional factors to transition to an active state.

RNA Polymerase Holoenzyme

• Definition: Composed of the core RNA polymerase and a sigma factor that facilitates transcription initiation.

• Function of Sigma Factor: Recognizes the promoter sequence to enable RNA polymerase to start transcription, ensuring the correct gene is expressed in response to cellular needs.

• Bacterial Sigma Factors Diversity: Different sigma factors regulate various genes in response to environmental signals, allowing bacteria to adapt quickly.

  • E. coli Examples:

    • σ70 (RpoD): Housekeeping sigma factor responsible for the expression of most genes under normal growth conditions.

    • σ38 (RpoS): Regulates genes during starvation and stress responses, facilitating survival in adverse conditions.

    • σ32 (RpoH): Activates heat shock genes to manage protein misfolding and stress.

    • σ54 (RpoN): Involved in nitrogen limitation, coordinating responses for effective nitrogen utilization.

Promoter Characteristics

• Definition: A DNA sequence that RNA polymerase binds to initiate transcription, crucial for determining the timing and level of gene expression.

• Identifying Methods: Techniques like DNA footprinting, sequence alignments, and mutagenesis studies are used for effective promoter localization in bacterial DNA, helping researchers understand regulatory mechanisms.

Mechanism of Prokaryotic Transcription

Stages:

• Initiation: Begins with RNA polymerase binding to the promoter region, forming a closed-promoter complex, followed by transition to an open-promoter complex where DNA is unwound.

• Elongation: RNA is synthesized during this phase as RNA polymerase moves along the DNA template, creating an RNA strand.

• Termination: The transcription process ends upon reaching a termination signal, which signals RNA polymerase to release the newly synthesized RNA molecule.

Transcription Initiation in E. coli

• Closed-promoter complex formation occurs with nonspecific binding of RNA polymerase to DNA, laying the groundwork for more precise transcription initiation.

• Transition to an open-promoter complex involves specific sigma factor binding to the -35 and -10 sequences, which leads to unwinding and initiation of RNA synthesis.

Transcription Elongation in E. coli

• The template strand is used for RNA synthesis, where RNA polymerase synthesizes RNA by forming phosphodiester bonds, adding NTPs to the growing chain.

• Synthesis proceeds in the 5’ to 3’ direction while maintaining the fidelity of the genetic information from the coding strand.

Transcription Termination in E. coli

• Elongation Ends: When RNA polymerase reaches a designated termination signal within the DNA, indicating the end of transcription.

• Termination Types:

  • Rho-independent: Involves formation of a GC-rich sequence with consecutive adenines, leading to a stem-loop structure that causes dissociation of RNA from DNA, concluding transcription rapidly.

  • Rho-dependent: Involves Rho protein that destabilizes the RNA-DNA interaction, promoting termination through a distinct mechanism of action.

Key Concepts

• Gene expression orchestrates multicellular development and cell differentiation, underscoring the importance of transcription in shaping organism functionality.

• The sigma factor of E. coli is crucial for RNA polymerase to bind to promoters, influencing gene expression patterns and responses to cellular environments.

• Termination of transcription can occur through hairpin formation or Rho factor mediation, highlighting the complexity of transcription regulation in prokaryotes.