RNA Transcription

DNA Synthesis and Repair

DNA Synthesis Steps

  • Newly synthesized DNA strands may have nicks in the phosphodiester backbone.

  • DNA ligase seals these nicks, completing the synthesis of daughter strands

DNA Repair Mechanisms

  • Mutations: Changes in the original DNA sequence.

    • Most mutations negatively affect protein function.

    • Some mutations have no effect on protein (silent mutations).

    • Rarely, mutations can enhance protein function.

  • Types of mutations:

    • Spontaneous mutations: Occur naturally during replication.

    • Induced mutations: Caused by external mutagens.

  • Cells have evolved several systems for DNA repair.

Single Base Mutations

  • Types of single base mutations:

    • Substitutions: One nucleotide exchanged for another.

      • Transition: Purine for purine or pyrimidine for pyrimidine.

      • Transversion: Purine for pyrimidine or vice versa.

    • Insertions: Addition of a nucleotide.

    • Deletions: Removal of a nucleotide.

  • Insertions or deletions can shift the reading frame affecting protein translation.

Classes of Single-Nucleotide Mutations

  • Silent mutations: No change in the amino acid sequence.

  • Missense mutations: Change in the coded amino acid.

  • Nonsense mutations: Change to a stop codon, terminating translation prematurely.

Mismatch Repair in E. coli

  • Nicked strand is degraded by an exonuclease and filled in by DNA polymerase III.

  • Mutations in human homologs of MutS & MutL can lead to susceptibility to colon cancer.

Base Excision Repair in E. coli

  • Maintains DNA integrity by removing incorrect bases (e.g. uracil) using DNA glycosylases.

  • Mechanism:

    • Nick is created, DNA polymerase removes and fills in the gap.

    • Finally, DNA ligase seals the nicks.

Double Strand Break Repair

  • Some DNA repair genes function as tumor suppressors.

  • BRCA1 gene is essential for repairing double-stranded breaks; mutations increase breast cancer risk significantly.

DNA Gel Electrophoresis

  • Process:

    • Melted agarose forms gel with comb to create slots.

    • DNA samples are loaded into the gel.

    • An electric current drives negatively charged DNA towards the positive pole (anode).

DNA Separation by Electrophoresis

  • DNA fragment size determined by movement through the gel.

    • Smaller fragments move faster, while larger fragments have more drag and move slower.

    • This creates a size gradient with larger fragments near the top.

    • Ethidium bromide used for staining; fluoresces under UV light.

DNA Size Estimation

  • Comparison with standard size markers allows estimation of fragment sizes.

  • Fragment mobility versus log of molecular weight plotted for analysis.

Central Dogma of Molecular Biology

  • Conceptual flow:

    • DNA replicates and is transcribed to RNA.

    • RNA is translated into protein sequences.

RNA Transcription and Processing

  • Transcription results in RNA having the same sequence as the DNA coding strand (T = U).

  • Genes can be transcribed from either DNA strand.

Transcription in Prokaryotes vs. Eukaryotes

  • Prokaryotic transcription is simpler and occurs in the cytoplasm, coupled with translation.

  • Eukaryotic transcription is more complex, occurring in the nucleus, with subsequent RNA processing.

Transcription Process

  • RNA polymerase synthesizes RNA in the 5’ to 3’ direction, without requiring primers.

  • Different subunits of RNA polymerase are involved depending on promoter specificity.

E. coli Transcription Unit

  • Elements of a transcription unit include:

    • Regions like -35 and -10, promoter, and terminator sequences.

Transcriptional Termination Mechanisms

  • Intrinsic termination: Forming a hairpin structure in RNA causes detachment.

  • Rho-dependent termination: Rho helicase facilitates termination by displacing RNA polymerase.

Eukaryotic mRNA Processing

  • Eukaryotic mRNAs undergo three processing steps in the nucleus:

    • Capping

    • Splicing

    • Addition of Poly(A) tail.

Capping of Eukaryotic mRNA

  • 5’ cap (N7-methyl G) protects mRNA from degradation and assists in ribosome recognition.

Splicing of Introns

  • Introns are non-coding sequences removed from mRNA during processing.

  • Self-splicing introns exist, but most splicing is done by the spliceosome.

Poly A Tail Addition

  • mRNA acquires a poly(A) tail to enhance stability and translation efficiency.

  • Recognized by the cleavage and poly(A) machinery at the AAUAAA consensus sequence.

Overview of Eukaryotic mRNA Processing

  • Gene transcription leads to primary RNA transcripts being processed into mature mRNA, including 5' capping, splicing, and 3' polyadenylation.

Translation Basics

  • Genetic information in mRNA is translated into amino acids.

  • Ribosomes and charged tRNA are essential for this process, facilitating the transition from nucleic acids to proteins.

tRNA Structure and Function

  • Charged tRNA acts as the adaptor, with an anticodon that pairs with mRNA codons for correct amino acid incorporation.

  • Each tRNA has a unique sequence but a common structural shape.

Ribosome Structure

  • Composed of two subunits made from RNA and proteins.

  • Eukaryotic ribosomes are larger than prokaryotic ones.

Translation Process

  • Translation involves three main steps:

    1. Initiation

    2. Elongation (decoding, peptide bond formation, translocation)

    3. Termination

Genetic Code Characteristics

  • Codons are triplets of nucleotides, non-overlapping, degenerate, and universal.

  • There are 61 codons for the 20 amino acids, plus 3 stop codons.

Example of Translation

  • Given an mRNA sequence, determine the corresponding one-letter amino acid code, indicating the start codon.

Elongation in Translation

  • Specifics of the elongation phase in protein synthesis, involving decoding the mRNA and forming peptide bonds.