HUBS1202 Human Genomics and Biomolecular Analysis Study Notes

HUBS1202 Human Genomics and Biomolecular Analysis

Overview

The course focuses on human genomics and biomolecular analysis. The primary instructor is Dr. Karen Mate from the School of Biomedical Sciences at The University of Newcastle, Australia.

Eukaryotic Gene Expression

  • Primary Transcript: Precursor to mature mRNA, termed pre-mRNA. It undergoes modifications at both ends, and introns are removed during maturation.

Transcription & Translation

  • Transcription occurs in the nucleus, where gene regions of DNA are transcribed into an RNA transcript.

  • Various RNA species include snRNA, rRNA, tRNA, mRNA, and pre-miRNA, which are further processed and translated in the cytoplasm.

  • Translation happens in the cytoplasm at ribosomes, resulting in a polypeptide chain.

  • Cytoplasmic pools: Contain amino acids, tRNAs, and ribosomal subunits crucial for translation.

  • miRNA represses mRNA translation.

Details on Transcription

  • Direction of Transcription: RNA is synthesized in the 5' to 3' direction using the DNA template strand. The coding (sense) strand is identical to the RNA transcript except that uracil (U) replaces thymine (T).

  • Example of Strand Orientation:

    • Template Strand: 3'-CGTATGCTAGTCCGATTGCG-5'

    • Nontemplate Strand (Coding): 5'-GCATACGATCAGGCTAACGC-3'

  • Transcription Bubble: Refers to the locally unwound segment of DNA where transcription occurs.

RNA Polymerases in Eukaryotes

  • RNA Polymerases: There are five types:

    1. RNA Polymerase I: Produces rRNA except for 5S rRNA, located in the nucleolus.

    2. RNA Polymerase II: Transcribes nuclear pre-mRNAs. Located in the nucleus.

    3. RNA Polymerase III: Synthetizes tRNAs and 5S rRNA. Located in the nucleus.

    4. RNA Polymerase IV: Found in plants, produces small interfering RNAs (siRNAs).

    5. RNA Polymerase V: Also found in plants, synthesizes noncoding transcripts of siRNA target genes.

  • RNA Synthesis: Catalyzed by RNA polymerases, occurring in three stages: initiation, elongation, and termination. It requires ribonucleoside triphosphates as precursors, using one strand of DNA as a template.

Initiation of Transcription

  • Initiation by RNA Polymerase II requires protein transcription factors such as TFIID, TFIIA, TFIIB, and TFIIF.

  • These proteins bind to the promoter region of the gene and facilitate the initiation of transcription.

RNA Polymerase II Promoter Structure

  • Key elements in a typical RNA Polymerase II promoter include:

    • TATA Box: Located about -25 upstream from the transcription start point.

    • GC Box and CAAT Box: Found in proximal control elements.

    • Core Promoter: Contains binding sites for transcription factors and RNA polymerase.

    • Transcription Start Point: Designated as +1.

RNA Processing

  • Pre-mRNA undergoes significant modifications:

    1. 5' Capping: Addition of a 7-methylguanylate (7-mG) cap to the 5' end to protect the mRNA and assist in ribosome binding at translation.

    2. 3' Polyadenylation: Addition of a poly(A) tail at the 3' end, which stabilizes the mRNA.

    3. Splicing: Removal of introns and joining of exons to form mature mRNA.

Excision of Intron Sequences

  • Introns have conserved dinucleotide sequences at the ends for precise removal during splicing. Typically, GU at the 5' splice site and AG at the 3' splice site.

Overview of Gene Expression

  • The transcription process translates DNA into RNA, producing rRNA precursors, mRNA, tRNAs, and other RNA products.

  • Translation thereafter generates polypeptides, utilizing transfer RNA (tRNA), ribosomal proteins, elongation factors, and GTP.

Translation

  • Codon Structure:

    • mRNA is read as triplet codons by ribosomes, initiating the translation of the encoded protein.

  • Initiation Codon (AUG): Marks the start of translation.

  • Triplet Code: Genetic code is composed of nucleotide triplets that are non-overlapping, degenerate, and nearly universal.

    • Example: The sequence AUC subsequently translates to isoleucine (Ile).

    • Stop Codons: Signal termination of the polypeptide chain. Includes UAA, UAG, and UGA.

Macromolecules of Translation

  • Components include polypeptides and RNA molecules of the ribosome, amino acid-activating enzymes, and tRNA molecules involved in the initiation, elongation, and termination of translation.

Recombinant DNA Techniques

  • Recombinant DNA: Consists of genetic material from multiple sources, producing sequences not typically found in a genome.

  • Gene Cloning: Involves isolation and amplification of a specific gene, forming a recombinant DNA molecule by joining different DNA segments.

Molecular Toolbox

  • Enzymes for DNA Manipulation:

    1. Restriction Endonucleases: Enzymes that cleave DNA at specific sequences (restriction sites), typically 4-8 base pairs long, creating reproducible fragments.

    2. DNA Ligase: Joins two DNA molecules or fragments by forming phosphodiester bonds.

    3. DNA Polymerase: Synthesizes new DNA strands by adding nucleotides.

Types of Cloning Vectors

Several cloning vectors allow the amplification of DNA of interest, notably:

  • Plasmids: Circular DNA that can accommodate up to 15 kb of inserted DNA.

  • BACs: Bacterial artificial chromosomes, capable of carrying up to 300 kb.

  • YACs: Yeast artificial chromosomes, with a maximum insert size of 600 kb.

Features of the Genetic Code

  • The genetic code consists of triplet codons, which are read without overlap, do not contain commas, and include start and stop signals facilitating the translation process.

  • Degeneracy refers to the ability of multiple codons to specify the same amino acid, which can lead to silent mutations.

  • The code is nearly universal, with a few variations in mitochondrial codons and in certain organisms.

Nucleic Acid Hybridization

  • Hybridization Process: Involves the pairing of complementary nucleic acid strands under controlled conditions, using techniques like Southern, Northern, and Western blotting.

    • Choose appropriate probes for specific DNA/RNA sequences to identify gene expression.

Key Points in Hybridization

  • The stability of hybrids is influenced by the number and type of base pairings. GC pairs form stronger interactions than AT pairs due to their additional hydrogen bond.

  • The melting temperature of the hybrid indicates the closeness of sequence similarity, with higher temperatures suggesting greater similarity between samples.