Gene Transcription Process

Promoter Elements

• The basal promoter of a gene consists of two types:
  • TATA box: Located 20-30 base pairs upstream of the transcription start site, binds basal transcription factors (TFs) and RNA polymerase II (RNAPII)
  • CCAT box: Located 50-130 base pairs away from the transcription start site, less common and weaker than the TATA box
• Promoter elements are essential for the transcription of all genes
  • They recruit RNAPII for the initiation of transcription

Transcription

• RNA Pol II transcribes just one strand of DNA (the template strand)
• RNAPII reads the template strand 3’ to 5’ and creates an RNA copy 5’ to 3’
• Transcription proceeds in 3 stages:
  • Initiation: RNAPII binds to the promoter and unwinds a 17-18 bp segment, forming the ‘Open Complex’
  • Elongation: RNAPII moves along the template strand, synthesizing RNA until it reaches the terminator region
  • Termination: Transcription continues beyond the protein-coding region, resulting in a 3’UTR (UnTranslated Region)
    • The Polyadenylation signal sequence (AAUAAA) within the transcribed sequence signals RNA Pol II to disengage
    • The nascent mRNA molecule is released for processing and degradation if necessary

mRNA Processing

• The RNA molecule transcribed by RNAPII is called the primary (1°) transcript
• Primary transcripts are also known as heterogenous nuclear RNA (hnRNA)
• hnRNA undergoes extensive modification
  • 5’ Capping: Addition of a 7-methylguanosine residue to the 5’ end of mRNA
    • The 5' cap protects mRNA from degradation, promotes export, and aids recognition by the translation machinery
  • 3’ poly(A) tail: Addition of a string of adenine residues (~40-250) to the cleaved 3’ end of mRNA
    • The poly(A) tail confers stability to mRNA molecules

Post-Transcriptional Regulation

• After transcription, a pre-mRNA molecule contains introns and exons
• Introns are non-coding, while exons encode the protein-coding region
• Pre-mRNA must undergo processing before translation
• Key areas of post-transcriptional regulation:
  • mRNA stability: The longevity of mRNA can be determined by specific sequences present in the 3’UTR
    • Sequences within the 3’UTR can target mRNAs for degradation
    • The 5’ cap protects mRNA from degradation
    • The poly(A) tail confers stability to mRNA molecules
  • Differential mRNA splicing: Specific combinations of exons are joined together, resulting in production of different protein isoforms from the same gene

Different Types of RNA

• mRNA: Messenger RNAs that code for proteins
• rRNA: Ribosomal RNAs, involved in protein synthesis and form the structure of the ribosome
• tRNA: Transfer RNAs, responsible for transferring amino acids to the ribosome during protein synthesis
• snRNA: Small nuclear RNAs, involved in a variety of nuclear processes, including pre-mRNA splicing
• ncRNA: Non-coding RNAs, regulate gene expression and participate in diverse cellular processes

Regulatory Non-coding DNA

• Formerly referred to as “junk DNA”
• The ENCODE (Encyclopaedia of DNA Elements) project revealed that a significant portion of the genome is actively transcribed and plays regulatory roles

miRNA

• Small non-coding RNA strands (20-25 nucleotides)
• Regulate gene expression by binding to complementary mRNA sequences
• Can either block translation or promote mRNA degradation
• Primarily act as negative regulators of gene expression

Inducible Transcriptional Regulation

• Cells have the ability to switch genes on or off in response to environmental cues, through the action of inducible transcription factors
• The activity of transcription factors is critical in controlling gene expression
• Transcription factors bind to specific DNA sequence elements and control the rate of transcription, helping to create a dynamic response to stimuli

Constitutive Gene Expression

• Some genes are constantly expressed, often encoding essential proteins involved in metabolism, repair, and essential cellular functions
• These genes are called constitutive genes due to their continuous expression

Inducible Gene Expression

• Inducible genes are only expressed when required, enabling responsiveness to environmental changes
• Induction of gene expression can be triggered by extracellular cues like hormones, cytokines, and cell-cell interactions
• Signals are transduced into the cell to initiate transcription of new genes, resulting in the synthesis of required proteins

RNA structure and function

• RNA is a nucleic acid similar to DNA, but with key differences
• RNA contains ribose sugar instead of deoxyribose
• RNA contains uracil (U) instead of thymine (T)
• RNA is single-stranded unlike DNA which is double-stranded.
• RNA plays a crucial role in gene expression by copying genetic information from DNA and contributing to ribosome formation.

Types of RNA

• mRNA (messenger RNA): carries genetic information from DNA to ribosomes for protein synthesis
• rRNA (ribosomal RNA): forms the structural and catalytic core of ribosomes, essential for protein synthesis
• tRNA (transfer RNA): acts as an adapter molecule during protein synthesis, carrying specific amino acids to the ribosomes based on the mRNA sequence
• snRNA (small nuclear RNA): involved in various nuclear processes, particularly pre-mRNA splicing
• ncRNA (non-coding RNA): a diverse group of RNA molecules that do not code for proteins, involved in various cellular processes: gene regulation, X-inactivation.

Transcription

• Transcription is the process of copying genetic information from DNA to RNA
• Transcription initiates at the Transcription Start Site (TSS)
• Promoters are DNA regions upstream of the TSS that regulate transcription initiation
• Two types of promoters: basal promoter elements and enhancer elements.
• Transcription ends at a termination sequence in the DNA.

Post-Transcriptional Processing

• Newly transcribed RNA molecules undergo post-transcriptional processing to become mature mRNAs.
• This process includes:
  • 5' capping: a modified guanine nucleotide is added to the 5' end of the mRNA, protecting it from degradation and aiding in ribosome binding.
  • 3' Polyadenylation: a string of adenine nucleotides (poly-A tail) is added to the 3' end of the mRNA, increasing its stability and facilitating export from the nucleus.
  • Splicing: non-coding introns are removed from the pre-mRNA, and coding exons are stitched together to form mature mRNA.

mRNA Stability

• The stability of mRNA molecules varies and is influenced by sequences within the mRNA, particularly in the 3' UTR.
• Destabilizing sequences in the 3' UTR can target mRNA for degradation by endonucleases.
• The 5' cap protects against degradation from exonucleases.
• The poly-A tail confers stability and is bound by poly-A binding protein (PABP), which protects the mRNA from degradation.

Splicing

• Splicing is the removal of non-coding introns from pre-mRNA
• Splicing is carried out by the spliceosome, a complex molecular machine composed of snRNAs and proteins.
• snRNPs (small nuclear ribonucleoproteins) are formed by the association of snRNAs with proteins.
• snRNPs recognize specific sequences at the ends of introns, cut the RNA at these sites and join the adjacent exons.

Inducible Gene Expression

• Inducible gene expression is controlled by transcription factors, which are proteins that regulate the rate of transcription of a gene.
• Inducible transcription factors are only active in response to specific stimuli.
• Transcription factors bind to specific DNA sequences, called response elements, and interact with RNA polymerase II to promote transcription.
• Some transcription factors can also recruit chromatin modifiers to aid in unwinding DNA or co-activators to enhance transcription.

Factors Affecting Transcription

• The number of binding sites for a specific transcription factor in a promoter determines the rate of transcription.
• Inducible transcription factors allow rapid and dynamic response to stimuli.

Basal Promoter Elements

• Essential for transcription of all genes.
• Recruits RNA Pol II for transcription.
• Binds basal transcription factors first, followed by RNA Pol II binding.
• Two main types:
  • TATA box: Most common, located 20-30 base pairs upstream of the transcriptional start site. Can bind basal transcription factors and RNA Pol II independently, making it a strong element.
  • CCAT box: Less common and weaker, located 50-130 base pairs upstream of the start site.

Transcription

• Catalyzed by RNA Polymerase II, transcribing one DNA strand from 3' to 5', creating an RNA copy from 5' to 3'.
• Incorporates ribonucleotide triphosphates (NTPs - A, G, C, & U) to build the mRNA copy.
• Three stages:
  • Initiation: RNA Pol II binds to DNA, unwinding a 17-18 bp segment of the promoter, forming the 'Open Complex'.
  • Elongation: RNA Pol II moves along the DNA template, synthesizing RNA until reaching the terminator region. A region of DNA under RNA Pol II remains unwound, creating the 'Transcription Bubble' that moves along the DNA.
  • Termination: Transcription continues past the protein-coding region into the 3'UTR (UnTranslated Region) forming a new mRNA molecule. Most eukaryotic mRNA precursors contain the motif 'AAUAAA' which:
    • Signals RNA Pol II to disengage.
  • Recruits an endonuclease to cleave the mRNA.
  • Poly(A) polymerase adds 40-250 adenine residues to the cleaved 3' end, forming the Poly(A) tail.

Post-Transcriptional mRNA Processing

• Primary transcript: RNA molecule synthesized by RNA Pol II.
• Heterogenous nuclear RNA (hnRNA): Collection of precursor molecules.
• Extensive modifications:
  • 5' capping: A 7-methyl-guanosine residue is added to the 5' end of the mRNA, creating an unusual 5' to 5' triphosphate linkage. This is catalyzed by guanylyltransferase (capping enzyme), followed by methylation by an ethyltransferase enzyme. The first and second nucleotides are also methylated.
  • Functions of 5' cap:
    • Protects mRNA from degradation by exonucleases.
    • Promotes nuclear export.
    • Aids recognition by translational machinery.
  • 3' poly(A) tail addition: After recognizing the polyadenylation signal sequence (AAUAAA), an endonuclease is recruited and cleaves the mRNA. Poly(A) polymerase then adds 40-250 adenine residues to the cleaved 3' end.

Post-Transcriptional Regulation: mRNA Processing

• Gene transcribes to produce mRNA containing introns and exons.
• Exons: Encode the protein coding region.
• Introns: Non-coding sequences removed during processing.
• Processing involves:
  • mRNA stability: 3' UTR determines mRNA stability, with destabilizing sequences in the 3' UTR being targeted for endonuclease degradation. The 5' cap protects against degradation, and exonucleases degrade from the poly(A) tail.
  • Differential mRNA splicing: Removal of introns while RNA is being synthesized, after capping but before transport to the cytoplasm.

Splicing of mRNA Precursors

• Introns are removed and adjacent exons are joined together.
• Introns range in size from 50 to ≥ 10,000 nucleotides.
• Spliceosome: Molecular machine carrying out splicing.
• Spliceosome components: Consists of RNA and protein.
• Small nuclear RNAs (snRNA): Combine with proteins to form small nuclear ribonucleoproteins (snRNPs), which facilitate splicing by recognizing and interacting with specific sequences at each end of the intron, cutting and rejoining the exons.
• Differential splicing/exon shuffling: Allows different combinations of exons to be joined, leading to the production of different proteins from the same gene, contributing to protein diversity.
• Examples:
  • Calcitonin processing: Produces calcitonin (thyroid) and cGRP (nerve).
  • Tropomyosin alternative splicing: Leads to the production of different isoforms of tropomyosin, contributing to muscle tissue diversity.

Importance of Gene Regulation

• All cells have the same DNA content but differentiate into distinct cell types.
• Precise regulation of gene expression is crucial to generate cell specificity by switching genes on and off.

Tissue Specificity

• Stem cells differentiate into various cell types, each displaying specific gene expression patterns, regulated by specific transcription factors.

Constitutive and Inducible Gene Expression

• Constitutive gene expression: Genes continuously expressed, such as those coding for metabolic or repair enzymes.
• Inducible gene expression: Genes expressed only when needed, allowing cells to respond to external signals.

Types of Genes

• Constitutive genes: Essential, continuously expressed.
• Inducible genes: Expressed only when needed, important for development and environmental response.
• Inducible gene expression is triggered by external cues:
  • Hormones: Estrogen, testosterone.
  • Cytokines: Interferons, growth factors.
  • Cell-cell interactions: Cell-matrix interactions.

Inducible Transcription Factors

• Necessary for controlling inducible gene expression.
• Only present or activated when necessary.
• Allow cells and tissues to respond to environmental cues.
• Example: Insulin transcription in pancreatic beta cells.

Transcription Factor Activity

• Determines the rate of transcription.
• Binds to specific DNA sequences called response elements.
• Interacts with RNA Pol II to facilitate transcription.
• Can recruit chromatin modifiers to aid in DNA unwinding.
• Can also recruit coactivators.
• Example: SP1 binding sites influence the rate of transcription by their number in a promoter.

Importance of Inducible Transcription Factors

• Allow for quick and dynamic responses to stimuli.
• Ensure only necessary genes are activated, contributing to efficient and relevant responses to changing conditions.

Explore the key elements of gene transcription, including the roles of the TATA and CCAT boxes as basal promoter components. This quiz covers the stages of transcription initiated by RNA polymerase II, detailing the process from initiation to termination. Test your knowledge of these essential molecular biology concepts.