BIOL212 W2-2

DNA and Genetic Information Flow

• The functional unit of genetic information is the gene.
• Genes are part of genetic elements, which are large molecules, such as chromosomes.
• Nucleic acids contain genetic information through nucleotides, which are monomers of nucleic acids.
• DNA is the genetic blueprint, and RNA is its transcription product.
• Both DNA and RNA are polynucleotides.
• Informational macromolecules include nucleic acids and proteins.

Genetic Elements: Chromosomes and Plasmids

• Genome size is expressed in base pairs.
  • 1000 base pairs = 1 kilobase pair = 1 kbp
  • 1 million base pairs = 1 megabase pair = 1 Mbp
• The E. coli genome is 4.64 Mbp and encodes around 4000 genes.
• Linear DNA is several hundred times longer than the cell, so supercoiling compacts DNA to accommodate the genome.
• Topoisomerases insert and remove supercoils.
• DNA gyrase introduces supercoils into DNA via double-strand breaks.
• Chromosomes are the main genetic element in prokaryotes.
• Most bacteria and archaea have a single circular chromosome carrying all or most of their genes.
• Plasmids are double-stranded DNA that replicate separately from the chromosome.
  • Usually circular.
  • Generally beneficial for the cell (e.g., antibiotic resistance).
  • Not extracellular, unlike viruses.
• Transposable elements are segments of DNA that can move from one site to another on the same or a different DNA molecule.
  • They can be inserted into other DNA molecules (e.g., chromosomes, plasmids, viral genomes).

Escherichia coli K-12 Chromosome Features

• Approximately 5 Mbp in size.
• GC content (% of G and C in the genome) is 50.8 %.
• Almost 4300 possible protein-encoding genes, comprising 88% of the genome.

Plasmid Features

• Plasmids can carry genes associated with virulence factors in pathogenic bacteria that contribute to infection.
• Bacteriocins, proteins that inhibit or kill closely related species or different strains of the same species, can be encoded on plasmids.
• Rhizobia use plasmid-encoded functions to fix nitrogen.
• Plasmids can contribute to metabolism, such as hydrocarbon degradation.
• Plasmids are crucial for conjugation, a form of horizontal gene transfer.

### DNA Replication

• DNA replication is semiconservative, meaning each new double helix consists of one original strand and one newly synthesized strand.
• Deoxynucleoside 5′-triphosphate (dNTP) serves as the precursor for each nucleotide.
• DNA replication always proceeds from the 5′ end to the 3′ end.
• DNA polymerases, specifically DNA Pol III, are the primary enzymes responsible for replicating chromosomal DNA in E. coli.
• Other DNA polymerases play roles in DNA repair.
• DNA synthesis initiates at the origin of replication in prokaryotes.
• DNA polymerases require a primer, a short stretch of RNA, which primase creates.
• The replication fork is the region of unwound DNA where replication occurs.
• DNA helicase unwinds the DNA.
• DNA synthesis occurs continuously on the leading strand and discontinuously on the lagging strand.
• Okazaki fragments are short segments of DNA synthesized on the lagging strand.
• DNA polymerase I removes the RNA primer and replaces it with DNA.
• DNA ligase seals the gaps in the DNA.
• Bidirectional replication occurs in circular chromosomes.
• Two replication forks move in opposite directions along the circular chromosome.
• DNA Pol III adds approximately 1,000 nucleotides per second.
• The replisome, a complex of multiple proteins involved in replication, consists of two copies of DNA polymerase III, DNA gyrase, helicase, primase, and multiple single-strand DNA-binding proteins.
• DNA replication is extremely accurate due to proofreading mechanisms that ensure high fidelity.
• Mutation rates in cells are typically 10–8 to 10–11 errors per base inserted.
• DNA Pol I and Pol III can detect mismatches through incorrect hydrogen bonding and remove them via their 3'→5' exonuclease activity.

RNA Synthesis: Transcription

• Central Dogma: DNA is duplicated (replication), information is transferred from DNA to RNA (transcription), and information in RNA is used to build polypeptides (translation).
• mRNA (messenger RNA) encodes proteins.
• tRNA (transfer RNA) converts mRNA into the amino acid sequence of a protein.
• rRNA (ribosomal RNA) provides catalytic and structural components for ribosomes.

### Transcription Characteristics in Prokaryotes & Eukaryotes

• Eukaryotes: Each gene is transcribed individually into a single mRNA. Replication and transcription happen in the nucleus. RNA must be exported outside the nucleus for translation.
• Prokaryotes: Multiple genes can be transcribed in a single mRNA. Transcription and translation are coupled, maximizing protein synthesis.

Transcription in Bacteria

• Transcription is the synthesis of RNA from a DNA template.
• RNA polymerase is the enzyme that carries out transcription.
• RNA polymerase requires DNA as a template and uses ATP, GTP, CTP, and UTP as RNA precursors.
• Transcription proceeds in the 5' to 3' direction, similar to DNA replication.
• Only one strand of DNA is transcribed.
• No primer is required for RNA transcription.

Prokaryotic vs. Eukaryotic RNA Polymerases

• Most eukaryotes have three different RNA polymerases:
  • Pol I: synthesizes ribosomal RNAs (rRNAs)
  • Pol II: synthesizes messenger RNAs (mRNAs)
  • Pol III: synthesizes 5S rRNA, transfer RNAs (tRNAs), and other small RNAs
• Plants have two additional RNA polymerases:
  • Pol IV: involved in the generation of small interfering RNAs (siRNAs) from transposons and repetitive sequences, contributing to epigenetic regulation.
  • Pol V: responsible for silencing (DNA and histone methylation) of transposons and repetitive sequences.
• Bacteria and Archaea only have a single RNA polymerase.

Bacterial RNA Polymerase Structure

• E. coli RNA polymerase is composed of five subunits: two α (αI and αII), β, β', and ω.
• The core enzyme consists of the β, β', and ω subunits.
• Together, the core enzyme and the sigma factor form the holoenzyme.

Key Features of Bacterial RNA Polymerase

• The DNA binding groove of RNA polymerase is positively charged, while DNA is negatively charged, facilitating the binding of DNA.
• Magnesium ions (Mg2+) are required for RNA polymerase activity.
• RNA polymerase lacks a proofreading mechanism, unlike DNA polymerase, which has 3' to 5' exonuclease activity.

Transcription Initiation in Bacteria

• Initiation is sequence-specific and occurs at the promoter sequence, located upstream of the RNA transcription start site.
• The core enzyme can catalyze transcription, but the sigma factor is needed for promoter recognition.
• Sigma factors are bacterial transcription factors that bind to the promoter sequence (-10 and -35 regions) and help RNA polymerase initiate transcription.
• The +1 site marks the transcription initiation site.
• The -10 region (also known as the Pribnow box) is a consensus sequence located about 10 base pairs upstream of the +1 site.
• The -35 region is another consensus sequence located about 35 base pairs upstream of the +1 site.
• Multiple sigma factors exist in bacteria, allowing RNA polymerase to recognize and transcribe different sets of genes.

Sigma Factor in E. coli

• E. coli has seven distinct sigma factors.
• The primary sigma factor, σ70, is responsible for the transcription of "housekeeping" genes.
• Each sigma factor typically binds to a set of genes with related functions, such as sporulation genes.

Examples of Sigma Factor Functions

• Sporulation sigma factor in Bacillus subtilis: responsible for transcribing genes involved in bacterial sporulation in response to adverse conditions.

RNA Polymerase Holoenzyme Structure

• The sigma factor is attached to the core RNA polymerase complex.

Anti-sigma Factors

• Inhibit sigma factor function by binding to them
• Regulate stress response in M.extorquens
• Phosphorylation of PhyR releases anti-sigma factor (NepR) from σEcfG1
• Allows σEcfG1 to associate with RNA polymerase and transcribe stress genes

Transcription Start and Elongation

• Closed Complex: DNA is double-stranded, RNA polymerase is bound to the promoter
• Open Complex: Sigma factor unwinds DNA at the promoter region, no helicase or ATP required
• Initiating Ribonucleotides: Transcription usually starts at +1 with a nucleotide triphosphate, resulting in a triphosphate at the 5' end of the newly synthesized RNA
• Elongation:
  • Sigma subunit detaches from the polymerase
  • Transcription bubble forms (18 base long DNA opening)
  • RNA/DNA hybrid forms, helping keep the polymerase attached to the DNA

Transcription Features

• Transcription can occur on either DNA strand
• Multiple RNA polymerases can transcribe the same gene simultaneously

Polycistronic mRNA

• Single mRNA molecule containing multiple genes
• Transcribed from the same promoter upstream of a cluster of genes with related functions (operon)

Ribosomal RNA

• Polycistronic

Coupled Transcription and Translation in Bacteria

• Ribosomes attach to the 5' end of mRNA before transcription is complete
• Ribosomes move along the mRNA towards the 3' end as transcription continues
• Allows protein synthesis to begin before mRNA is fully transcribed
• Not possible in eukaryotes due to separate compartments for transcription and translation

Transcription Termination

• RNA polymerase detaches from the template when it encounters a terminator sequence
• Two types: factor-independent and Rho-dependent

Factor-Independent Termination

• Inverted repeat sequence with a central non-repeating segment forms a hairpin structure
• Followed by a run of U nucleotides
• Destabilizes RNA/DNA binding, leading to RNA dissociation

Rho-Dependent Termination

• Requires Rho factor - an RNA-dependent ATPase and RNA/DNA helicase
• Recognizes rut sites in RNA
• Rho binds to rut and acts as a helicase, moving 5' to 3' along the transcript
• This pulls apart the RNA/DNA hybrid, releasing the RNA polymerase when it's paused at the terminator sequence

Anti-sigma Factors

• Anti-sigma factors are proteins that bind to sigma factors, inhibiting their function.
• Phosphorylation of PhyR in Mycobacterium extorquens triggers the release of the anti-sigma factor NepR from the sigma factor σEcfG1.
• This release allows σEcfG1 to associate with RNA polymerase, leading to transcription of genes involved in stress response.

Transcription Initiation and Elongation

• Closed Complex: RNA polymerase bound to the promoter region, DNA is double-stranded.
• Open Complex: DNA unwinds at the promoter region, catalyzed by the sigma factor.
• Initiating Ribonucleotides: Transcription starts at the +1 site with a nucleotide triphosphate, creating a triphosphate at the 5' end of RNA.
• Elongation: Sigma subunit dissociates, forming a transcription bubble with an 18 base pair RNA/DNA hybrid.
• Transcription Features: Transcription can occur in either DNA strand depending on the gene, and multiple RNA polymerases can transcribe a single gene simultaneously.

Polycistronic mRNA

• One mRNA molecule contains multiple genes transcribed from the same promoter upstream of a gene cluster.
• These clusters, called operons, usually contain genes with related functions.

Coupled Transcription and Translation in Bacteria

• Ribosomes attach to the 5' end of mRNA even before transcription is complete.
• Ribosomes move along the mRNA towards the 3' end as transcription continues.
• This coupling allows protein synthesis to start before mRNA is fully produced, reducing the delay between transcription and protein expression.

Transcription Termination

• Factor-Independent Termination:
  • Inverted repeat sequence (high G+C content) forms a hairpin structure through intramolecular base-pairing.
  • Run of U residues following the inverted repeat destabilizes RNA/DNA binding, leading to RNA dissociation.
• Rho-Dependent Termination:
  • Rho protein is an RNA-dependent ATPase and RNA/DNA helicase.
  • Rho binds to rut (rho utilization) sequences in RNA, moving along the transcript in a 5' to 3' direction.
  • Rho unwinds the RNA/DNA hybrid, causing RNA polymerase to disengage when paused at the terminator sequence.