Comprehensive Notes on Transcription and Translation

Elongation

  • Elongation is a phase of transcription.
  • Key components involved:
    • RNA polymerase
    • Non-template strand of DNA
    • RNA nucleotides
    • DNA and RNA molecules
    • Specific nucleotide sequences (ATCCAA, CAU, CCA, GGT)

Central Dogma

  • Gene expression occurs in two steps:
    1. Transcription
    2. Translation
  • Central dogma of information flow: DNA \rightarrow RNA \rightarrow Protein
  • Transcription involves transcribing DNA-based genetic information into RNA, maintaining the language of nucleotides.

Types of RNA Molecules

  • mRNA (messenger RNA): Carries genetic information from DNA to ribosomes.
  • tRNA (transfer RNA): Involved in transporting amino acids to the ribosome for protein synthesis.
  • rRNA (ribosomal RNA): A component of ribosomes, crucial for protein synthesis.
  • snRNS (small nuclear RNA): Participates in splicing and spliceosome formation.
  • snoRNS (small nucleolar RNA): Involved in maturation of pre-rRNA, pre-tRNA, and snRNA molecules.
  • microRNA: Short, single-stranded non-coding RNAs that integrate into RISC, affecting mRNA translation or degradation, leading to gene silencing.
  • siRNS: Double-stranded RNA molecules processed into RISC, causing mRNA degradation and gene silencing.

Replication vs. Transcription

  • Replication: Duplication of entire DNA double helix.
  • Transcription: mRNA transcription from a template strand in the region of genes.
  • Complementarity: Similar to DNA replication, except uracil (U) replaces thymine (T) in RNA.
  • Transcription speed: Approximately 40 times slower than replication.

Key Aspects of Transcription

  • Transcription produces single-stranded RNA complementary to one strand of DNA.
  • RNA synthesis is facilitated by DNA-dependent RNA polymerase.
  • RNA synthesis does not require primers.
  • Helicases and topoisomerases are not needed; RNA polymerase manages helix unwinding and rewinding.
  • Synthesis direction: 5’ to 3’.
  • Start and stop signals in DNA indicate where RNA polymerase should begin and end transcription.

Coding and Template Strands

  • Coding strand: Base sequence matches that of the RNA molecule.
  • Template strand: Serves as a template for transcription.

Gene Location and RNA Polymerase

  • Genes can be found on either strand of DNA.
  • RNA polymerase unwinds and rewinds DNA during transcription.

RNA Polymerase

  • Possesses 5’ \rightarrow 3’ polymerase activity.
  • Lacks proofreading activity.
  • E. coli has one type of RNA polymerase: holoenzyme = core enzyme (\alpha_2, \beta, \beta’ subunits for elongation) + \sigma (sigma) factor (promoter recognition).
  • Eukaryotic cells have three types of RNA polymerase:
    • RNA polymerase I: Transcribes rRNA genes.
    • RNA polymerase II: Transcribes protein-coding genes and snRNAs.
    • RNA polymerase III: Transcribes tRNA and 5S rRNA genes.

Biosynthesis Stages

  • Initiation: Starting the process (involves the \sigma -factor).
  • Elongation: Chain extension.
  • Termination: Ending the process.

Transcription Unit

  • The segment of DNA transcribed into RNA, spanning from the promoter to the terminator.
  • Longer than the region between the translational START and STOP codons.

Promoter Elements

  • Typical prokaryotic promoter has three elements:
    • -35 sequence.
    • -10 sequence (TATA box or Pribnow box).
    • Transcription start point.
  • Translation start point follows the transcription start point; in prokaryotes, it’s found after the ribosome binding site (Shine-Dalgarno sequence) on the mRNA.
  • Promoter is responsible for transcription initiation and serves as the RNA polymerase binding site.
  • RNA polymerase:
    • First interacts with the -35 region, forming a closed complex with DNA.
    • Then interacts with the -10 region, transitioning from a closed to an open complex.

Promoter Types

  • Constitutive promoters: For genes transcribed continuously (e.g., tRNA, rRNA, ribosomal proteins, glycolysis enzymes).
  • Inducible promoters: For genes transcribed only under specific conditions.

Prokaryotic Transcription and Translation

  • Prokaryotic transcription is coupled with translation, resulting in monocistronic mRNA.
  • Genes in operons are transcribed into polycistronic mRNA, encoding multiple proteins.
  • Features include:
    • 5’ and 3’ untranslated regions.
    • Coding region.
    • Ribosome binding sites/Shine-Dalgarno sequences.
    • Rare standalone genes.

RNA Synthesis

  • RNA synthesis proceeds in the 5’ \rightarrow 3’ direction.
  • RNA polymerase adds nucleotides complementary to the template DNA strand.

Transcription Bubble

  • RNA synthesis takes place within a transcription bubble, where DNA strands separate.
  • One strand serves as the template for RNA synthesis.

Elongation Phase

  • RNA synthesis begins at the start site and continues until the stop site.
  • Involves RNA polymerase, template strand, growing RNA strand, and release of the sigma factor upon termination.

Termination Types

  • Rho-independent: GC-rich signal sequence forms a hairpin structure, causing RNA polymerase to stall and the RNA transcript to detach.
  • Rho-dependent: Requires Rho factor to separate the DNA-RNA hybrid when RNA polymerase pauses at the termination sequence.

Rho Factor and Ribosomes

  • Interaction between Rho factor and ribosomes can occur in nonsense mutations.

Eukaryotic Gene Structure

  • Regulatory sequences: Binding sites for specific proteins.
  • Exon: Protein-coding region.
  • Intron: Non-coding region.
  • Gene-proximal regulatory sequences.
  • Transcription start point: 5’UTR.
  • Cap site.
  • Poly-A signal: Termination signal.

Eukaryotic Transcription Initiation

  • General transcription factors bind to DNA and each other in a specific order.
  • TFIID binds to the TATA box, altering DNA structure to allow further transcription factor binding.
  • TATA box is a consensus sequence in most eukaryotic promoters, binding general transcription factors.
  • Initiation in eukaryotes is more complex than in prokaryotes.
  • Requires general and specific transcription factors for RNA polymerase II binding and transcription initiation.

Polymerase Activation

  • Involves sequence-specific DNA-protein interactions and protein-protein interactions.
  • Cooperative interactions among specific activators, general transcription factors, and the mediator complex.

Elongation in Eukaryotes

  • RNA polymerase moves along the DNA template, adding nucleotides to the growing RNA strand.
  • Nucleotides are added to the 3’ end (5’ \rightarrow 3’ growth).
  • Transcription speed: 40 nt/sec.

Termination and Polyadenylation in Eukaryotes

  • A poly-A signal (AAUAAA) is transcribed into RNA.
  • Endonuclease binds to the poly-A signal and cleaves the mRNA.
  • Poly-A polymerase synthesizes a 100-300 nucleotide poly-A tail at the 3’ end.
  • Transcription continues past the termination sequence, with the pre-mRNA’s 3’ end later cleaved and polyadenylated.

Pre-mRNA Processing

  • Capping: Addition of a methylated guanine to the 5’ end.
  • Splicing: Removal of introns.
  • Polyadenylation: Addition of a poly-A tail to the 3’ end.
  • Eukaryotes: pre-mRNA undergoes modification.
  • Prokaryotes: mRNA does not undergo modification; transcription and translation are coupled. Mature mRNA is produced immediately.

5’ Cap Structure

  • Addition of a 7-methylguanylate molecule (cap) to the 5’ end of hnRNA.
  • Cap stabilizes mRNA and is needed for translation, maturation, and transport to the cytoplasm.

Splicing

  • Occurs via spliceosomes (intron-excising complexes).
  • Involves a lariat-like structure.
  • Invariants important in introns.

Poly-A Tail Formation

  • Enzymatic process catalyzed by poly(A)-polymerase (PAP).
  • Requires other proteins (CPSF, CStF, CFI, CFII, PABPII).
  • Consensus sequence: AAUAAAxxxxxxxG/Uxxx.

Role of Cap and Poly-A Tail

  • Cap: Involved in translation initiation, protection from nucleases.
  • Poly-A tail: Required for mRNA transport from the nucleus to the cytoplasm, increases mRNA lifespan, signals ribosomes.

rRNA and tRNA Processing

  • rRNA and tRNA molecules are produced through primary transcript maturation in all cell types.

Central Dogma

  • DNA replication, repair, and genetic recombination.
  • RNA synthesis (transcription).
  • Protein synthesis (translation).

Genome, Transcriptome, Proteome, Metabolome, and Interactome

  • Genome: Complete description of genes in an organism.
  • Transcriptome: Complete description of mRNA in a genome.
  • Proteome: Complete description of proteins expressed by a genome.
  • Metabolome: Complete description of metabolites expressed by a genome.
  • Interactome: Catalog of all protein-DNA, protein-RNA, and protein-protein interactions.

Gene Expression Levels

  • Vary depending on the rate of transcription and translation.

Translation Overview

  • 3 nucleotides in a gene = 1 triplet.
  • 3 nucleotides on mRNA = 1 codon.
  • 1 amino acid in a protein.
  • Conversion from DNA/RNA language to protein language.

Amino Acids

  • The building blocks of proteins.
  • 20 different amino acids.

Genetic Code

  • Codon: A triplet of bases on mRNA.
  • 64 codons: 61 code for amino acids, 3 are stop codons.
  • Genetic code is:
    • Universal.
    • Degenerate.
    • Unambiguous.

Transfer RNA (tRNA)

  • tRNAs are 74-95 nucleotides long, with a characteristic cloverleaf shape.
  • Different tRNAs transport different amino acids.
  • 30-50 different tRNAs per cell.
  • Aminoacyl-tRNA synthetase attaches the correct amino acid to the tRNA molecule.
  • Anticodon: A triplet of bases complementary to the mRNA codon.

Wobble Hypothesis

  • Third base of the mRNA codon and the corresponding tRNA pairing may not strictly adhere to complementarity rules.
  • Different codons specifying the same amino acid (differing only in the 3rd base) are often recognized by the same tRNA molecule.

Aminoacyl-tRNA Synthetases

  • Responsible for linking tRNA molecules to their corresponding amino acids.
  • Among the most specific enzymes in the cell.

Mitochondrial Genetic Code

  • Differs from the universal genetic code.
  • Human mitochondrial genome code differs from that of plants and fungi.

Translation Key Concepts

  • Ribosomes.
  • Codon: mRNA base triplets that specify amino acids or stop signals.
  • Anticodon: tRNA base triplets that bind to mRNA codons.
  • Aminoacyl-tRNA synthetase: Attaches the correct amino acid to the correct tRNA.
  • Peptidyl transferase activity: rRNA catalyzes peptide bond formation.
  • START codon: Where translation begins (AUG).
  • STOP codon: Where translation ends (UAG, UAA, UGA).
  • ORF (Open Reading Frame): Begins with a START codon and ends with a STOP codon.

Ribosome Binding Site

  • Shine-Dalgarno sequence.

Ribosomes

  • Composed of large and small ribosomal subunits.
  • Have A, P, and E sites.

Ribosome Composition

  • Prokaryotic and eukaryotic ribosomes differ in their rRNA and tRNA components.
  • Eukaryotic ribosomes contain 28S, 18S, and 5.8S rRNAs encoded by acrocentric chromosomes; 5S rRNA is encoded by chromosome 1.

Translation Mechanism

  • Initiation: Ribosome and initiator tRNA bind to mRNA at the START codon.
  • Elongation: Ribosome moves along mRNA, adding amino acids to the growing polypeptide chain.
  • Termination: Translation ends when a STOP codon is reached.

Prokaryotic Initiation

  • Small ribosomal subunit binds to mRNA at the Shine-Dalgarno sequence.
  • Initiator tRNA binds to the first AUG codon.
  • Large subunit binds.

Eukaryotic Initiation

  • Initiator tRNA (Met) binds to the small ribosomal subunit.
  • Small subunit recognizes the mRNA cap region and scans for the first AUG codon.
  • Large subunit binds.

Elongation Process

  • Aminoacyl-tRNA corresponding to the next codon enters the A-site.
  • Peptide bond forms between the amino acid in the A-site and the polypeptide in the P-site.
  • Ribosome translocates one codon along the mRNA.

Termination Process

  • STOP codon (UAG, UAA, UGA) in the A-site is recognized by termination factors.
  • Polypeptide is released from the tRNA, and the ribosome dissociates.

Polyribosomes (Polysomes)

  • Multiple ribosomes simultaneously translate a single mRNA, forming a polyribosome.

Eukaryotic Gene Expression

  • Transcription and translation are spatially and temporally separated.
  • Protein synthesis occurs after mRNA processing and transport to the cytoplasm.
  • One mRNA molecule encodes one protein (monocistronic).

Alternative Splicing

  • One gene can produce multiple proteins through alternative promoter usage, alternative splicing, and alternative polyadenylation.

Genetic Code Evolution

  • The genetic code is organized to minimize the effects of point mutations.

Bacterial Translation Inhibition

  • Some antibiotics selectively inhibit translation in bacteria.
  • Examples:
    • Streptomycin: Inhibits initiation.
    • Tetracycline: Inhibits tRNA binding.
    • Erythromycin: Inhibits translocation.
  • Side effects occur because mitochondria have prokaryotic-type ribosomes.