Prokaryotic Genomes and Gene Structures

• RNA vaccines use a lipid coat for delivery, enabling fusion with the cell membrane.
• RNA must be stable to enter cells without degradation.
• Polymerase is encoded with a spike protein transcript for rapid amplification inside the cell.

Prokaryotes

• Plasmids contain non-essential DNA, such as antibiotic resistance genes.
• Plasmids replicate independently and might integrate into eukaryotic chromosomes.
• Prokaryotes multiply via binary fission.
• The prokaryotic genome consists of 4 million base pairs in a circular, supercoiled structure.
• DNA replication initiates from a single origin of replication.
• Intergenic regions are non-gene regions.
• Transcription and translation occur simultaneously.
• Prokaryotes are mostly haploid, with one copy of each gene.
• Genes are organized in operons, allowing polycistronic transcription.

Prokaryote Gene Structure

• Genes encoding repressors or transcription factors are located separately.
• Operons are regulated by multiple factors.
• Eukaryotes favor separate genes on separate strands for cell specialization.
• Prokaryotes prioritize replication efficiency.
• Mutations at the beginning of an operon can affect downstream genes.
• Synchronized timing of gene expression is an advantage

Trp Operon

• The Trp operon is anabolic.
• Tryptophan is essential for protein biosynthesis.
• Features a strong promoter.
• It is regulated by a repressor protein.
• High tryptophan levels inhibit expression by activating the repressor.

Lac Operon Components

• This operon encodes enzymes for lactose breakdown.
• It exhibits beta-galactosidase activity.
• LACZ encodes beta-galactosidase, splitting lactose into galactose and glucose.
• LACY encodes a transporter protein facilitating lactose transport across the cell membrane.
• LACA encodes transacetylase, detoxifying toxic lactose analogues.
• Lac Z and Lac Y are positive control elements and break down lactose

Lac Operon Function

• The Lac operon has a medium-strength promoter requiring a repressor and transcription factor.
• The repressor is always bound unless allolactose is present.
• Low glucose levels lead to high cAMP levels, activating the CAP transcription factor.

Secondary Structures of RNA

• tRNA anticodon stem loop enables codon recognition during translation
• rRNA in ribosomes is composed of 50% protein and 50% RNA.
• RNA forms 3D structures due to its single-stranded nature and self-base pairing.

Transcription Initiation

• Polymerase binding to single-stranded DNA requires a transcription bubble.
• Promoter consensus sequences are essential.
• The exact promoter sequence determines its strength.
• The promoter indicates the polymerase binding site.
• Strand direction determines transcription direction (always 5' to 3').
• Polymerase consists of two alpha subunits, two beta subunits, and one omega subunit.
• The sigma factor is vital for recognizing and driving expression.
• An apoprotein/apoenzyme is a polymerase not bound to a cofactor.
• A holoenzyme/holoprotein is a polymerase bound to a cofactor.

Initiation Steps

• Sigma associates with the core holoenzyme, binding at -35 and -10 consensus sequences.
• The holoenzyme binds to the promoter and unwinds DNA.
• Energy for bond formation comes from NTP hydrolysis.
• Bond formation occurs between nucleoside triphosphates.
• Topoisomerase relieves torsional strain.
• Sigma is released after the polymerase moves away from the consensus sequence.
• RNA polymerase must release its hold on the promoter to advance downstream

Elongation Steps

• Promoter clearance is when RNA polymerase breaks free of the sigma factor.
• Core RNA polymerase adds rNTPs (ribosomal nucleotides) to grow the chain.
• rNTPs are added using energy from NTP hydrolysis, releasing two phosphates.
• DNA unwinds ahead of RNA polymerase and rewinds as it moves forward.
• Topoisomerases relieve torsional strain.
• Limited proofreading occurs.
• Elongation continues until a termination signal is reached.

Termination Steps: Rho-Independent

• RNA forms hairpin structures, displacing polymerase and halting transcription, usually due to inverted repeats.
• Base pairing occurs in inverted repeats to stop transcription.
• Long stretches of As and Us create instability, promoting secondary structures.

Termination: Rho-Dependent

• The Rho protein dissociates the polymerase from the template.
• Rho recognizes a specific terminator site called RUT.
• The RUT site is an unsaturated region.
• NusG connects Rho to RNA polymerase, enhancing termination.
• RNA polymerase stalls upon encountering a terminator sequence.
• Rho is always present and binds to mRNA at the RUT site.
• After binding, Rho catches up to the stalled polymerase.

Genetic Code

• Prokaryotes often use overlapping reading frames in their operons.
• Redundancy exists, with multiple codons coding for the same amino acid.
• Silent mutations occur in the third position of a codon.
• Missense mutations occur in the first or second position.
• Nonsense mutations change an amino acid to a stop codon.

Types of RNA

• Messenger RNA (mRNA) carries information copied from DNA.
• Transfer RNA (tRNA) deciphers the code and delivers amino acids.
  • The acceptor stem is where the amino acid joins.
  • The anticodon loop matches the codon via base pairing.
• Ribosomal RNA (rRNA) associates with proteins to form ribosomes.

Protein Synthesis RNA

• Enzymes that attach amino acids to tRNA are amino acid transferase synthetases.

Phases of Translation

• Charging of RNA
• Initiation
• Elongation
• Termination

Charging of RNA

• It describes how amino acids attach to the acceptor stem of tRNA:
  • A high-energy intermediate forms between cAMP and the amino acid.
  • Energy stored in the intermediate attaches the amino acid to the tRNA.
  • A covalent bond forms.
  • Hydrolysis of the bond releases energy for polypeptide chain growth.
• tRNA folding variations aid in attaching specific amino acids.
• Each tRNA synthetase recognizes multiple tRNAs.
• One tRNA can recognize multiple mRNA codons due to wobble.
  • Wobble is flexibility in the last base of a codon in mRNA.
• tRNA synthetase ensures correct binding using anticodon recognition and proofreading.

Initiation

• IF3 prevents the large subunit from binding to the small subunit.
• The small subunit recognizes the Shine-Dalgarno sequence upstream of the start codon.
  • This sequence aligns the ribosome correctly.
• The first tRNA binds to AUG (methionine) at the P site.
• In prokaryotes, methionine is post-translationally modified with a formal charge.
  • Formal methionine (fMet) marks the beginning of the code.
• After translation, the formal charge is removed.
• IF3 is removed, and the large ribosomal subunit binds.

Elongation

• Elongation factors, including EF-Tu, bind the aminoacyl-tRNA to the ribosome.
  • EF-Tu, a G protein, binds GTP and facilitates aa-tRNA binding to the A site.
• IF3 helps large and small ribosomal subunits separate for mRNA reformation.
• IF2 helps place fMet in the correct ribosome position.

Steps

• fMET enters the P site; IF2 helps place it.
• Elongation begins with the full ribosome complex.
• EF-Tu binds and guides the aminoacyl-tRNA complex to the A site.
• EF-Tu hydrolyzes GTP to GDP, releasing EF-Tu and allowing tRNA entry.
• 23S rRNA catalyzes peptide bond formation between amino acids in the A and P sites.
• EF-G binds and hydrolyzes GTP, pulling mRNA through the ribosome.
• Proteins form from the N-terminus onward.
• Empty tRNA is recycled.

Antibiotics

• Target transcription and translation in prokaryotes (bacteria).

Translation Termination

• RF1 or RF2 attach to the ribosome's A site, binding RF3-GTP.
• GTP is hydrolyzed, releasing tRNA, mRNA, and RF.
• RF1, RF2, and RF3 are release factors that stall ribosomes at stop codons.

Lac Operon Regulation

• The lac operon, with a medium-strength promoter, needs a transcription factor and a repressor.
• It is only needed in the presence of lactose.
  • Allolactose binds to the repressor, allowing transcription.
  • Allolactose is produced by beta-galactosidase.
• Glucose negatively regulates the lac operon.
  • With the absence of glucose cAMP is produced.
  • cAMP binds to CAP proteins, activating them as transcription factors.

Gene Regulation General Notes

• Weak promoter is default off; needs a transcription factor but not a repressor.
• Strong promoter is default on; needs a repressor but not a transcription factor.
• Positive control involves transcription factor addition or removal.
• Negative control involves repressor protein addition or removal.

LacI vs LacO

• LacI is the gene encoding the repressor.
• LacO is the operator sequence (DNA).

Operons

• Catabolic operons break down molecules (e.g., Lac).
  • Glucose is a catabolite of lactose metabolism.
  • Catabolite repression involves glucose decreasing cAMP and repressing CAP.
  • CAP is a catabolite activator protein, with a ligand-binding domain and a DNA-binding domain.
• Anabolic operons encode biosynthetic pathways (e.g., Trp).

Trp Operon

• It has conditional repression.
• It has a strong promoter and two repressors.
  • Attenuation
  • Trp repressor
• It is on when Trp is low, off when Trp is high.

Attenuation

• Attenuation is an interaction between transcription and translation
  • Termination happens when sections 3 and 4 bind.
• When Trp is high, polymerase does not stall, and section 3 binds with section 4 which leads to termination of transcription.
• When Trp is scarce, polymerase stalls over Trp codons, allowing section 2 to bind. This helps prevent attenuation and allows normal translation.
• Regulatory Trp codons must be further up on the 5' end of the operon.
• Attenuation works only in prokaryotes where translation and transcription occur simultaneously.
• The Trp repressor is more efficient but both sense different Trp concentrations.
  • Very high or low Trp = repressor protein needed
  • Medium Trp amounts = attenuation needed

Complementation

• Plasmid complementation introduces a wild-type gene copy into a mutated organism.
• Plasmids use their own promoters so it can be expressed regardless if the operon is on or shut off.

Trans Acting

• A physical protein like a repressor protein that affects multiple copies of genes
• If a mutated repressor protein needs a functional repressor protein to return to normal function

Cis Acting

• A DNA regulatory element that can only regulate the strand it is on
• If the DNA regulatory element has mutated, adding a copy would not fix the strand.