Molecular Biology and Biochemistry - Transcription

The Central Dogma

  • DNA contains information in a sequence of bases.
  • Proteins are made from a sequence of amino acids.
  • RNA acts as an intermediate 'messenger' by making a copy of the DNA.
  • Only certain bits of the DNA are copied, and multiple copies are made.
  • Proteins perform the work in a cell.
  • The central dogma states that once information gets into a protein, it can’t get out again.

Flow of Genetic Information

  • Perpetuation of genetic information from generation to generation.
  • Control of the phenotype through gene expression.
  • Replication: DNA-dependent DNA polymerase
  • Transcription: DNA-dependent RNA polymerase, resulting in mRNA
  • Translation: Complex process involving ribosomes, tRNAs, and other molecules, resulting in a polypeptide
  • Reverse transcription: RNA-dependent DNA polymerase (reverse transcriptase)

Gene Expression

  • Prokaryotic transcription occurs without a nucleus and involves simpler regulation.
  • Regulation is crucial in gene expression because all cells contain the same information, but only specific genes are transcribed and translated in particular cells.
  • Muscle cells express more actin and myosin.
  • Kidney cells express more ion transporters.
  • Neuron cells express more ion channels and neurotransmitters.
  • All cells express common genes for metabolic pathways, cell receptors, and membrane assembly.

Regulation of Gene Expression

  • Regulation occurs at various stages:
    • DNA: Replication, Degradation
    • RNA: Transcription (Initiation, Elongation, Processing, Export), Degradation
    • Protein: Translation (Initiation, Elongation, Termination), Targeting

Prokaryotic vs Eukaryotic Transcription

  • Prokaryotic Transcription
    • No nucleus
    • The primary transcript is equivalent to the mature mRNA.
    • Codons on the mRNA are translated into an amino acid sequence by ribosomes.
  • Eukaryotic Transcription
    • With a nucleus
    • The primary transcript is pre-mature (pre-mRNA).
    • The pre-mRNA is modified at both ends, and introns are removed to produce the mRNA.
    • After processing, the mature mRNA is exported to the cytoplasm for translation by ribosomes.

Timing of Transcription and Translation

  • Prokaryotes: Transcription and translation are coupled; proteins are synthesized directly from the primary transcript as it is made.
  • Eukaryotes: Transcription and translation are separated; transcription occurs in the nucleus, and translation occurs in the cytoplasm on ribosomes.

Cellular Location of Transcription and Translation

  • Prokaryotes: Transcription and translation occur in the cytoplasm.
  • Eukaryotes: Transcription occurs in the nucleus, and translation occurs in the cytoplasm.

Types of RNA and Their Function

  • mRNA: Encodes the sequence of amino acids.
  • tRNA: Transfer RNA; charged with amino acids, serves as a link between mRNA and the growing amino acids chain during translation.
  • rRNA: Ribosomal RNA; components of ribosomes, important for translation.
  • microRNA: Short RNA involved in gene regulation.
  • scRNA: Small cytoplasmic RNA; important for protein secretion in bacteria and protein targeting to the ER in eukaryotes.
  • RNA in RNaseP: Ribozymes of this enzyme.
  • snRNA: Small nuclear RNA; important in splicing.
  • Telomerase RNA: Template for reverse transcription.
  • snoRNA: Small nucleolar RNA; RNA processing in the nucleus.
  • Viral RNA: Genome of the virus.

General Features of RNA Polymerization

  • Similar to DNA polymerization except:
    • Substrates are ribonucleoside triphosphates: ATP, GTP, CTP, UTP (dNTP in DNA replication).
    • Only one strand of DNA serves as a template (compared with both DNA strands in DNA replication).
    • RNA polymerization can be initiated de novo (no primer required).
  • The RNA molecule will be:
    • Complementary to the DNA template (antisense) strand.
    • Identical (except that base uridine replaces thymidine) to the DNA non-template (sense) strand.
  • RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5’ → 3’ direction.

Template and Coding Strands

  • The template or antisense (-) strand is complementary to the sequence of the RNA transcript.
  • The coding or sense (+) strand of DNA has the same sequence as the transcript, with T in place of U.
  • DNA synthesis is antiparallel and occurs in the 5’ to 3’ direction.

RNA Polymerases

  • Catalyze the nucleophilic attack of the 3’-hydroxyl group of the last nucleotide in the chain on the α phosphoryl group of the incoming nucleoside triphosphate.
  • Elongation occurs when a ribonucleoside triphosphate base-pairs with a nucleotide on the DNA template.
  • The 3′-hydroxyl group of the last nucleotide in the chain attacks the α-phosphoryl group of the incoming nucleoside triphosphate, reforming a phosphodiester linkage and releasing PPiPP_i (pyrophosphate).

RNA Chains

  • Formed de novo and grow in the 5′-to-3′ direction.
  • The 5′ end of the transcript is usually pppG or pppA.

Transcription in Prokaryotes

  • Polymerization catalyzed by RNA polymerase, which can initiate synthesis, uses rNTPs, requires a template, and unwinds and rewinds DNA.
  • Three stages:
    • Initiation (Recognition and binding)
    • Elongation
    • Termination and release

Regulatory Sequences on DNA

  • Regulatory sequences: Sites for the binding of regulatory proteins; these influence the rate of transcription. They can be found in a variety of locations.
  • Promoter: Site for RNA polymerase binding; signals the beginning of transcription.
  • Terminator: Signals the end of transcription.

Stages of Transcription

  • Initiation: The promoter functions as a recognition site for transcription factors, which enable RNA polymerase to bind to the promoter. Following binding, the DNA is denatured into an open complex.
  • Elongation/synthesis of the RNA transcript: RNA polymerase slides along the DNA in an open complex to synthesize RNA.
  • Termination: A terminator is reached that causes RNA polymerase and the RNA transcript to dissociate from the DNA.

Initiation: Formation of Transcription Bubble

  • Binding of RNA polymerase holoenzyme to a promoter region in DNA.
  • Localized unwinding of the two strands of DNA by RNA polymerase to provide a single-stranded template.
  • Formation of phosphodiester bonds between the first few ribonucleotides in the nascent RNA chain.
  • RNA synthesis occurs in a complex called the transcription bubble, where approximately 17 bases of the DNA are unwound.
  • For another nucleotide to become incorporated into the transcript, translocation of RNA polymerase must occur. The transcription bubble must move to allow the next nucleotide to base-pair with the DNA template.
  • As the bubble moves, the RNA product exits the enzyme, and the transcribed DNA rejoins its partner.

Starting Transcription

  • Specific sequences signify the beginning of a gene; the promoter region (compared with the origin in DNA replication).
  • The first nucleotide to be transcribed is denoted as +1. The nucleotide immediately preceding it is denoted as –1.

Promoter Recognition

  • Located on the sense strand/coding strand/non-template strand.
  • Promoters function to provide a stable binding site for RNA polymerase and transcription factors.
  • Core promoter elements for E. coli include the -10 box (Pribnow box) and the -35 box.
  • RNA polymerase binds to different promoters with different strengths; binding strength relates to the level of gene expression.

The Promoter

  • Contains a consensus sequence; not all promoters have this exact sequence, but the nearer they are to it, the more strongly RNA polymerase binds to it.
  • The promoter can be on either DNA strand; whichever one it is on, the opposite (template) strand is transcribed.
  • Both strands of DNA can act as the template in different sections.
  • The promoter region specifies the site and direction of mRNA synthesis.

DNA Strands as Templates

  • Both DNA strands can be templates but synthesize different RNA transcripts and peptides.
  • The promoter region specifies the starting site and direction of transcription.

Typical Prokaryote Promoter

  • The transcript initiation site is +1.
  • Pribnow box (TATAAT) located at –10.
  • -35 box.
  • Consensus sequences (Non-template strand).

Common Features of E. Coli Promoters

  • Strong promoters are more similar to the consensus sequence; weak promoters have more variations.
  • Changes in position and sequence of -10 element and changes in the distance between the -10 and -35 sequences affect efficacy.

E. Coli RNA Polymerase

  • Multisubunit:
    • Core enzyme (5 subunits): α2ββ’ωα_2ββ’ω
    • Holoenzyme = Core (α,α,β,β’,ωα, α, β, β’, ω) + sigma (σ)
  • Functions of the subunits:
    • α: Holds enzymes together.
    • β: Ribonucleoside triphosphate binding site.
    • β’: DNA template binding region.
    • ω: Maintains the conformation and recruits β’.
    • σ: DNA recognition, unwinds DNA, initiation of transcription.
  • Transcription starts at a promoter sequence and ends at a terminator sequence.

Sigma (σ) Factor

  • Essential for recognition of promoter.
  • RNA polymerase only binds to specific sequences (promoters) with tight affinity when the sigma factor joins it to form the RNA polymerase holoenzyme, stimulating transcription.
  • Combines with core → holoenzyme, adopting an “open hand” conformation and positions the enzyme over the promoter.
  • Does NOT stimulate elongation (it blocks the movement of RNA polymerase).
  • Falls off after 4-9 nucleotides are incorporated, then the “hand” closes.

Initiation of Transcription Steps

  • Formation of closed promoter (binary) complex.
  • Formation of open promoter (binary) complex.
  • Ternary complex (RNA, DNA, and enzyme), abortive initiation - the polymerase creates short mRNA transcripts that are released before the polymerase detaches itself from the promoter.
  • Promoter clearance (elongation ternary complex):
    • First ribonucleotide (rnt) becomes unpaired.
    • Polymerase loses sigma.
  • Elongation starts - ribonucleotides added to 3’ end.

Promoter Clearance and Elongation

  • Occurs after 4 - 10 nucleotides are added.
  • The first ribonucleotide becomes unpaired from the antisense (template) strand, and DNA strands re-anneal.
  • Polymerase loses sigma, which is recycled, resulting in a “closed hand” that surrounds DNA.
  • NusA binds to core polymerase to stabilize the binding.
  • The transcription bubble moves along the DNA as DNA is unwound and then rewound, while the RNA product is extruded from the complex.
  • RNA pol/NusA complex stays on until termination. Rate=20-50 nt/second.
  • NO PROOFREADING - Unlike DNA polymerase, RNA polymerase has no 3’ to 5’ exonuclease activity, resulting in 1 mistake in 10,000 nucleotides.

Transcription Bubble

  • RNA polymerase unwinds and rewinds DNA.
  • Template strand is used for synthesis, and the coding strand is displaced.
  • Nascent RNA is synthesized with an RNA-DNA hybrid helix.

Termination

  • Occurs at specific sites on the template strand called Terminators.
  • Two types of termination:
    • Rho (ρ)-independent/ intrinsic terminator
    • Rho (ρ) dependent terminators

Rho-Independent Termination

  • Contains inverted complementary sequences that form a hairpin when transcribed, which slows transcription.
  • A second repeat sequence is polyA (polyU on RNA), which is weak, and the transcript separates from the DNA template.
  • Transcription terminates when inverted repeats form a hairpin followed by a string of uracils.

Characteristics of Rho-Independent Termination

  • Transcription is terminated due to a specific sequence in the terminator DNA.
  • The polymerase reaches a termination sequence of guanines and cytosines, followed by a sequence of repeating adenines.
  • A loop is formed in the guanine-cytosine region, and as guanine forms three hydrogen bonds with cytosine, it takes longer for the RNA Polymerase to join these.
  • This puts a strain on the adenine region and causes the strand to break off, releasing the polymerase.
  • RNA polymerase passes over inverted repeats, hairpins begin to form in the transcript, and the poly-U:poly-A stretch melts, causing RNA polymerase and transcript to fall off.

Rho-Independent Termination Mechanism

  • Stem-loop structure causes RNA polymerase to pause.
  • U-rich sequence is not able to hold the RNA-DNA hybrid together.
  • Termination occurs.

Rho-Dependent Termination

  • Rho factor is an ATP-dependent helicase.
  • Rho binds to a particular sequence on the RNA (called the rho utilization site, rut) and uses the energy of ATP hydrolysis to chase down the polymerase in the transcription bubble.
  • Contact with rho causes the transcription bubble to dissociate, catalyzing unwinding of the RNA: DNA hybrid.
  • Rate: 50~90 nucleotides/sec.

Characteristics of Rho-Dependent Termination

  • Rho factor protein binds to regions with no secondary structure.
  • The RNA sequence upstream from termination doesn't form secondary structure.
  • Rho factor binds to RNA and moves toward the 3' end.
  • At a hairpin, transcription slows, and rho factor can