Transcription and Its Control

Transcription

  • Transcription is the first step in the flow of genetic information from DNA to protein.
  • It's the initial stage of gene expression, where a DNA nucleotide sequence (gene) is transcribed into an RNA nucleotide sequence.
  • This process is called transcription because the information in a nucleotide sequence (DNA) is copied to another nucleotide sequence (RNA).

DNA and RNA sequences

  • DNA consists of two strands with base pairs.
  • RNA is synthesized using one strand of the DNA as a template for complementary base pairing.
  • mRNA has the same sequence as the upper strand of the DNA (sense sequence) and is complementary to the lower strand of the DNA (antisense sequence).

Gene Expression

  • Genes can be expressed with different efficiencies, leading to varying amounts of RNA and protein products.

RNA molecule

  • RNA (Ribonucleic acid) is a single-stranded polynucleotide chain.
  • It consists of ribonucleotides bound by phosphodiester bonds.
  • RNA contains ribose as its sugar and the bases adenine, guanine, cytosine, and uracil.

RNA Polymerases

  • RNA polymerases are the enzymes that catalyze transcription.
  • RNA polymerase does not require a primer to initiate synthesis.
  • The enzyme proceeds step by step along the DNA, opening the double helix.
  • It uses one DNA strand as a template, adding nucleotides to the growing RNA chain.
  • The nucleotides are added in the form of ribonucleoside triphosphates, and the energy from the phosphate-phosphate linkage fuels the polymerization.

Types of RNA Polymerases in Eukaryotes

  • Eukaryotic cells have three types of RNA polymerase:
    • RNA polymerase I: transcribes 5.8S, 18S, and 28S rRNA genes.
    • RNA polymerase II: transcribes all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, lncRNA genes, and most snRNA genes.
    • RNA polymerase III: transcribes tRNA genes, 5S rRNA genes, some snRNA genes, and genes for other small RNAs.
    • rRNAs are named according to their “S” values, which relate to their rate of sedimentation in an ultracentrifuge; larger S values indicate larger rRNAs.

DNA sequences and Transcription Factors

  • Transcription factors bind to specific DNA sequences to regulate transcription.
  • Common elements include:
    • BRE (TFIIB recognition element).
    • TATA box (recognized by TBP, a subunit of TFIID).
    • INR (Initiator element, recognized by TFIID).
    • DPE (Downstream promoter element, recognized by TFIID).

Transcription factors

  • Transcription factors are proteins required for the initiation of transcription but are not subunits of RNA polymerase.

General Transcription Factors

  • Bind to specific DNA sequences.
  • Ensure RNA polymerase is correctly located at the promoter region.
  • Determine the starting region of transcription.
  • Help separate the two DNA strands to allow RNA polymerase to proceed to elongation.

Initiation of Transcription by RNA Polymerase II

  • Requires additional proteins for in vivo transcription.
  • Gene-regulating proteins (transcriptional activators) help RNA polymerase collect at the transcription initiation site by binding to specific DNA sequences.
  • Adaptor proteins mediate the interaction between activators and RNA polymerase II, as well as general transcription factors.
  • Chromatin remodeling enzymes modify the DNA to make it accessible for transcription.

Steps for Eukaryotic Gene Transcription Initiation by RNA Polymerase II

  • Transcription factors bind to the promoter region in a specific order: TFIID, TFIIB, TFIIE, TFIIH.
  • TFIID binds to the TATA box with its TBP subunit.
  • TFIIB, TFIIE, TFIIH, and TFIIF, along with RNA polymerase, then bind to form the transcription initiation complex.
  • TFIIH hydrolyzes ATP to phosphorylate RNA polymerase II's CTD (carboxy-terminal domain) and open the DNA double strand.
  • Phosphorylation of the CTD causes general transcription factors to be replaced by RNA-modifying enzymes.

Accessory Proteins

  • Accessory proteins are required for the elongation of transcription.
  • RNA polymerase can pause or accelerate at specific sequences.
  • Elongation factors interact with RNA polymerase to prevent its separation from the template DNA.
  • ATP-dependent chromatin remodeling enzymes facilitate the movement of RNA polymerase along the gene.
  • Histone chaperones reassemble histones behind the RNA polymerase after their separation from the DNA.

Primary RNA Transcript Modification

  • Primary RNA transcript undergoes three main modifications:
    • Addition of a 5’ cap.
    • RNA splicing.
    • Addition of a Poly-A tail.

RNA Modification during Transcription

  • RNA-modifying enzymes are carried by the DNA polymerase and transferred to the newly formed RNA molecule at the appropriate times during transcription.

5’ Cap Addition

  • Addition of the 5’ cap is carried out by three enzymes:
    • Phosphatase: removes a phosphate group from the 5’ end of the RNA.
    • Guanyl transferase: adds a GMP to the RNA with a 5’-5’ bond.
    • Methyl transferase: adds a methyl group to guanosine.
  • This is the first reaction in eukaryotic pre-mRNA processing.

Functions of the 5’-Methyl Cap

  • Marks the 5’ end of eukaryotic RNA, distinguishing mRNA from other RNA molecules.
  • In the nucleus, the cap binds to a protein complex, which aids in further mRNA modification and transport to the cytoplasm.
  • Involved in protein translation.

Gene Structure: Exons and Introns

  • Human genes, such as the human β-globin gene and human Factor VIII gene, contain arrangements of exons (coding regions) and introns (non-coding regions).

RNA Splicing

  • RNA splicing is the removal of non-coding intron sequences from precursor mRNA transcripts.

Spliceosome

  • Reactions in RNA splicing occur via a complex called the spliceosome.
  • The spliceosome complex consists of small nuclear RNA molecules (snRNAs) and their associated proteins (U1, U2, U4, U5 and U6).
  • Each splicing event requires the hydrolysis of several ATP molecules.

Sequences Indicating Intron Removal

  • Sequences that indicate the regions to be removed during splicing include:
    • Branching point.
    • Pyrimidine-rich region (~15 bases).
    • Exon Joining region

SR Proteins and Exon Identification

  • SR proteins bind to exonic splicing enhancers (ESEs) in exons and are important for identifying exons in large pre-mRNAs.
  • An interaction network between SR proteins, snRNPs, and splicing factors forms a cross-exon recognition complex that determines the correct splicing regions.

RNA Splicing Process

  • RNA splicing removes intron sequences from newly synthesized RNA molecules.
  • Each splicing event removes one intron.
  • The pre-mRNA splicing reaction occurs as follows:
    • A specific adenine nucleotide in the intron sequence attacks the 5’ splice region, breaking the sugar-phosphate backbone of the RNA.
    • The spliced 5’ end of the intron binds covalently to the adenine.

Esterification Reactions in Splicing

  • First esterification: the 3’ oxygen of Exon 1 reacts with the 5' splice site.
  • Second esterification: the 3’ oxygen of the Exon 2 reacts with the 5' end of Exon 1, joining the exons and releasing the intron as a lariat.

Spliceosome Mechanism

  • Step 1: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point.
  • Step 2: U4/U6/U5 snRNP complex binds, displacing U1.
  • Step 3: First esterification: U2 and U6 catalyze the attack of the branch point A on the 5' splice site, forming a lariat.
  • Step 4: Second esterification: U5 aligns the exons for ligation.
  • Result: Combined exons and lariat intron are released. The lariat is then debranched by a debranching enzyme, yielding a linear intron RNA.

Alternative Splicing

  • Alternative splicing allows the encoding of different proteins from a single mRNA.
  • Example: α-tropomyosin gene can be spliced in various ways to produce different mRNAs in striated muscle, smooth muscle, fibroblasts, and brain.

Dscam Gene Splicing

  • The Drosophila Dscam gene undergoes extensive alternative splicing of its RNA transcripts.
  • It can produce one out of 38,016 possible splicing patterns.

Significance of RNA Splicing

  • RNA splicing enhances the coding capacity of the genome.

Splicing Errors

  • Two types of splicing errors include:
    • Exon skipping.
    • Cryptic splice-site selection.

mRNA 3’ End Processing

  • The 3’ end of each mRNA molecule is determined by signals present in the genome.
  • After RNA polymerase II transcribes these signal sequences, specific proteins and enzymes that process RNA bind to these recognition sequences.
  • CstF (Cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor) are important proteins involved in the formation and processing of the 3’ end of mRNA.

Poly-A Tail Addition

  • CstF and CPSF proteins bind to their specific sequences as soon as they are synthesized in the RNA transcript.
  • The synthesized transcript is separated from the RNA polymerase.
  • Poly-A Polymerase adds about 200 adenine nucleotides to the 3’ end of the transcript.
  • Proteins bound to the tail determine the length of the poly-A tail through an as yet not fully understood mechanism.

Nucleosome interference

  • Transcription regulator binding can be affected by nucleosome positioning.
    • A typical transcription regulator will bind with 20 times lower affinity if its cis-regulatory sequence is located near the end of a nucleosome.
    • A typical transcription regulator will bind with roughly 200-fold less affinity if its cis-regulatory sequence is located in the middle of a nucleosome.
  • One transcription regulator can destabilize the nucleosome, facilitating binding of another.

Chromatin Structure Alterations

  • Eukaryotic transcription activator proteins direct local alterations in chromatin structure through:
    • Chromatin remodeling.
    • Nucleosome removal.
    • Histone replacement.
    • Histone modification.
  • These alterations allow access of transcription machinery to DNA and destabilize compact forms of chromatin.

Repression of Transcription

  • Ways of repression of transcription of eukaryotic transcriptional repressors:
    • Recruitment of chromatin remodeling complexes.
    • Recruitment of histone deacetylases.
    • Recruitment of histone methyl transferases.

Transcription Regulators Binding

  • Transcription regulators bind sequence-specifically to DNA and regulate transcription.
  • Each transcription regulator contacts the DNA (mostly the major groove) via hydrogen bonds, ionic bonds, and hydrophobic interactions.

Transcriptional Regulator

  • Transcriptional regulators attach to specific sequences as monomers, dimers, or heterodimers.
  • Dimerization increases their specificity and affinity for DNA.

Gene Control Region

  • The gene control region for a typical eukaryotic gene includes:
    • General transcription factors.
    • RNA polymerase II.
    • Transcription regulators.
    • Coactivators
    • The promoter and cis-regulatory sequences

Eukaryotic Transcription Regulators

  • Eukaryotic transcription regulators assemble into complexes on DNA.
  • These complexes can either activate or repress transcription.

Transcriptional Activators

  • Transcriptional activators exhibit transcriptional synergy, leading to enhanced transcription.

Repression of Transcription Mechanisms

  • Ways of repression of transcription of eukaryotic transcriptional repressors:
    • Competitive DNA binding.
    • Masking the activation surface.
    • Direct interaction with the general transcription factors.