Gene Function: Transcription Notes

Gene, Chromosome, Genome

  • Genome: The complete set of genes or genetic material present in a cell or organism, including both the coding and non-coding regions. It provides a comprehensive blueprint for the structure and function of an organism.

  • Chromosome: A thread-like structure of nucleic acids and protein found in the nucleus of most living cells. Chromosomes are composed of chromatin, which is a complex of DNA and proteins. They carry genetic information in the form of genes, arranged linearly along the DNA molecule.

  • Gene: A locus (or region) of DNA, which is made up of nucleotides and is the molecular unit of heredity. Genes contain the instructions for building specific proteins or performing specific functions in the cell. Each gene has a specific location on a chromosome.

  • Chromosome number varies dramatically between species. Examples:

    • Common fruit fly: 88

    • Human: 4646

    • Dog: 7878

    • Kingfisher: 132132

Gene Structure & the Central Dogma

  • Eukaryotic Gene Structure:

    • Promoter Region: A regulatory region upstream of the gene, containing the TATA box and other regulatory elements that control gene expression.

    • Exons: Coding regions of the gene that contain the information to produce a protein. Exons are interspersed with non-coding regions called introns (Exon1, Exon2, Exon3).

    • Introns: Non-coding regions within a gene that are transcribed into RNA but are removed by splicing during RNA processing (Intron1, Intron2).

    • Transcription Initiation Site: The specific location on the DNA where transcription begins, often marked by a Cap sequence.

    • Translation Initiation Site: The site where translation begins, typically identified by the codon ATG, which codes for methionine.

    • Transcription Termination Site: The sequence of DNA that signals the end of transcription.

    • Translation Termination Site: The codon (TAA, TGA, TAG) that signals the end of translation, causing the ribosome to release the newly synthesized protein.

    • Poly A Site: The site where the poly-A tail is added to the mRNA molecule, signaling the end of transcription.

    • Enhancers: Regulatory sequences that can increase transcription levels.

  • Central Dogma:

    • DNA is transcribed into RNA: The process by which the information in DNA is copied into RNA.

    • RNA is translated into protein: The process by which the information in RNA is used to synthesize proteins.

    • Nuclear RNA is processed into Messenger RNA (mRNA): Pre-mRNA molecules are processed to remove introns and add a 5' cap and a 3' poly-A tail.

    • mRNA is then translated into protein to form Protein Complexes: mRNA carries the genetic code from the nucleus to the ribosomes, where it is translated into a protein.

Transcription

RNA Synthesis

  • RNA Polymerases are protein complexes with 8-14 subunits, responsible for synthesizing RNA molecules from a DNA template.

  • Three types of RNA polymerases in Eukaryotes:

    • Pol I: Transcribes ribosomal RNAs (rRNAs), which are essential components of ribosomes.

    • Pol II: Transcribes mRNA for translation into protein. It also transcribes small nuclear RNAs (snRNAs) involved in splicing.

    • Pol III: Transcribes small RNAs (tRNAs), which are used to carry amino acids to the ribosome during translation. It also transcribes other small RNAs.

  • RNA synthesis occurs in a specific direction, from the 5' end to the 3' end, using the template strand as a guide.

  • The coding strand and template strand are involved: The coding strand has the same sequence as the RNA transcript (except T is replaced by U), while the template strand is complementary to the RNA transcript.

A. Initiation

  • Transcriptional initiation is a major way of regulating gene expression, determining when and where a gene is transcribed.

  • Estimated number of genes:

    • Human: ≈25,000−30,000\approx 25,000 - 30,000

    • Round Worm: ≈19,000\approx 19,000

    • Yeast: ≈6,000\approx 6,000

  • At any given time, only a fraction of these genes are actively transcribed, depending on the needs of the cell or organism.

  • Transcriptional Initiation Process:

    • RNA polymerase holoenzyme binds to the promoter region on the DNA, along with other proteins known as transcription factors.

    • General transcription factors bind to the eukaryotic promoter, helping to position RNA polymerase and initiate transcription.

    • TBP (TATA-binding protein) binds to the TATA box, a specific DNA sequence within the promoter region, and helps to unwind the DNA.

    • Preinitiation complex is formed: A complex of transcription factors and RNA polymerase that assembles at the promoter.

    • RNA polymerase II begins elongation, synthesizing the RNA molecule.

  • Key Promoter Elements

    • BRE: TFIIB Recognition Element (-37 to -32): A sequence recognized by the TFIIB transcription factor.

    • TATA Box (-31 to -26): TATAAA: A conserved DNA sequence that is bound by the TATA-binding protein (TBP).

    • Initiator Element (Inr) (-2 to +4): Dm: TCAGTT, Hs: PyPyANT PyPy: A sequence that defines the transcription start site.

    • Downstream Promoter Element (DPE) (+28 to +32): GGG CCACGCC AGATA: A sequence found in some promoters that lack a TATA box.

Transcription Factors

  • General transcription factors and gene regulatory proteins are involved: These proteins help to regulate the rate of transcription.

  • Transcription factors bind to regulatory sequences of the gene, such as enhancers and silencers, to modulate transcription.

  • Transcription factors mediate the targeting of basal transcription factors and RNA polymerase to the promoter, ensuring that transcription is initiated at the correct location.

  • Role of Transcription Factors:

    • Activators and co-activators enhance transcription by increasing the rate of RNA synthesis.

    • Repressors inhibit transcription by decreasing the rate of RNA synthesis.

  • Transcription factors recognize and bind to bases without disrupting base pairing, using specific DNA-binding domains.

  • Transcription Factor Domains:

    • Trans-activating domain: Activates transcription by interacting with other proteins.

    • Protein Interaction: Facilitates interactions with other transcription factors or regulatory proteins.

    • DNA Binding: Binds to specific DNA sequences in the promoter or enhancer regions.

  • Multiple DNA Binding Motifs in Transcription Factors:

    • Helix-Turn-Helix: A common DNA-binding motif found in many regulatory proteins.

    • Zinc Finger: A DNA-binding motif that uses zinc ions to stabilize its structure.

    • Leucine Zipper: A DNA-binding motif that forms a coiled-coil structure with leucine residues.

    • Helix-Loop-Helix: A DNA-binding motif that consists of two alpha helices connected by a loop.

  • Helix-Turn-Helix motif fits into the major groove of DNA, allowing the transcription factor to interact with specific DNA sequences.

B. Elongation

  • RNA polymerase moves along the template strand, unwinding DNA and synthesizing RNA.

  • NTPs (nucleotide triphosphates) are added to the 3' end of the RNA molecule, extending the RNA chain.

  • A transcription bubble is formed with unwound DNA of approximately 1818 bp, allowing RNA polymerase to access the template strand.

  • RNA/DNA hybrid region is approximately 1212 bp: A short region where the newly synthesized RNA is base-paired with the DNA template.

  • Topoisomerases are required to manage the unwinding of DNA, preventing supercoiling and tangling of the DNA molecule.

  • Rudder and Zipper are structural elements involved in elongation, helping to guide the RNA transcript and maintain the stability of the transcription complex.

C. mRNA processing & Termination

  • Occurs after transcription: After the RNA molecule has been synthesized, it undergoes several processing steps before it can be translated into protein.

  • Steps:

    • 5’ Cap Addition: Covalent addition of a methylated G to the 5' end to protect from degradation and initiate protein synthesis. This cap also helps the ribosome bind to the mRNA.

    • Splicing: Removal of introns, which are non-coding regions, and joining together of exons, which are coding regions.

    • 3’ Poly A Tail Addition: Addition of 40-200 adenine nucleotides to the 3’ end for stability and to facilitate exit of the mRNA from the nucleus. The poly-A tail also helps to protect the mRNA from degradation and enhance translation.

    • Termination: The process by which transcription is ended and the RNA polymerase is released from the DNA template.

  • 5' Cap Addition Details:

    • 7-methyl guanosine triphosphate cap: A modified guanine nucleotide that is added to the 5' end of the mRNA molecule.

    • Protects from degradation: The cap protects the mRNA from being degraded by enzymes in the cell.

    • Initiates protein synthesis: The cap helps the ribosome bind to the mRNA and initiate translation.

  • 3’ Polyadenylation Details:

    • Poly-A tail is added after transcription by poly-A polymerase: The poly-A tail is added to the 3' end of the mRNA molecule by an enzyme called poly-A polymerase.

    • CPSF (Cleavage and polyadenylation Specificity Factor) is involved: CPSF is a protein that recognizes the poly-A signal sequence on the mRNA and recruits poly-A polymerase.

    • PAP (Poly A Polymerase) adds the poly-A tail: Poly-A polymerase adds adenine nucleotides to the 3' end of the mRNA molecule, forming the poly-A tail.

    • Xm2 nuclease degrades remaining nascent transcript, leading to termination of transcription: After the poly-A tail has been added, an enzyme called Xm2 nuclease degrades the remaining RNA transcript, leading to termination of transcription.

  • mRNA Splicing:

    • Introns are removed, and exons are joined together: Introns are non-coding regions of the gene that are removed from the pre-mRNA molecule by splicing. Exons are coding regions of the gene that are joined together to form the mature mRNA molecule.

    • Splicing occurs in the nucleus: Splicing takes place in the nucleus, where the pre-mRNA molecule is located.

  • Alternative Splicing:

    • Allows for the production of different proteins from a single gene: Alternative splicing allows different combinations of exons to be included in the mature mRNA molecule, resulting in the production of different protein isoforms.

    • Tissue-specific splicing is common (e.g., a-tropomyosin): Alternative splicing can occur in a tissue-specific manner, resulting in the production of different protein isoforms in different tissues.

    • Splicing mechanism: pre-mRNA -> lariat structure formation -> exons joining: Splicing involves the formation of a lariat structure, in which the intron is looped out and the exons are joined together.

Summary of Transcription

  1. Initiation: RNA polymerase and transcription factors bind to the promoter, initiating transcription.

  2. Elongation: RNA polymerase