Molecular Biology: Transcription Process

Cystic Fibrosis and DNA Editing

• CFTR is a chloride ion channel
• Mutations in the CFTR gene lead to non-functional CFTR
• Non-functional CFTR results in no chloride ion transport
• Improper hydration of airways and intestines is a consequence

"Mini-guts" or Organoids

• Organoids are miniature gut structures
• Functional CFTR can be activated by a chemical called forskolin

CRISPR Editing

• CRISPR can be used to correct the CFTR F508Del mutation
• Cells can be taken from a patient's intestine for editing
• CRISPR-edited cells can be compared to unedited cells to assess the effect

DNA Editing in Cystic Fibrosis

• CFTR F508Del is a mutation in CF patients
• Patient intestinal cells can be collected and edited by CRISPR
• Edited cells (F508Del-Corrected clone) are compared to non-edited cells

Genetic Engineering of Humans

• Articles mention the prospect of genetic enhancement
• The Economist, MIT Technology Review, and The Spectator discuss human genome engineering

Gene Expression

• The process of transcription converts DNA to RNA
• The process of translation converts RNA to proteins
• Non-coding RNA includes tRNA and rRNA, which are transcribed from specific regions on DNA

Structure of RNA and DNA

• Ribose sugar is in RNA, and deoxyribose is in DNA
• Uracil is in RNA, and thymine is in DNA
• DNA is more stable than RNA

RNA World Hypothesis

• Many scientists believe the first nucleic acids were RNA molecules
• RNA is involved in numerous cellular processes (including steps in the central dogma)
• RNA also possesses enzymatic properties
• DNA's stability may be the reason cells use it over RNA

RNA vs. DNA

• RNA has ribose sugar, DNA has deoxyribose sugar
• RNA has uracil, DNA has thymine
• RNA is single-stranded, DNA is double-stranded
• RNA is generally smaller than DNA

Transcription

• DNA serves as the template for RNA production
• Transcription occurs similarly in prokaryotes and eukaryotes, but in different locations
• Transcription is initiated at a promoter and ends at a terminator
• The product is synthesized in a 5' to 3' direction

Initiation of Transcription

• DNA molecules frequently contain numerous genes
• Transcription begins at a promoter and ends at a terminator sequence
• RNA polymerase and associated proteins bind to the DNA duplex at the promoter
• The synthesized product is RNA in a 5' to 3' direction
• Which strand is transcribed may vary from gene to gene

Promoter Recognition in Eukaryotes

• Promoters are complex
• Enhancers located close to or far from a gene
• General transcription factors and activator proteins
• A general transcription factor TBP binds to the TATA box

RNA Polymerase and Elongation

• During elongation, RNA polymerase adds nucleotides according to the template strand
• The 3' hydroxyl group of the growing RNA strand attacks the high-energy phosphate group of the incoming nucleoside triphosphate, which provides the energy for polymerization

RNA Polymerase in Prokaryotes

• RNA Polymerase in prokaryotes has distinct channels for nucleotides entry, transcript exit, and DNA template binding.

Primary Transcript in Prokaryotes and Eukaryotes

• In prokaryotes, transcription and translation occur together.
• In eukaryotes, primary transcripts undergo processing before translation
• Polycistronic mRNA contains information from multiple genes

5' Cap of Eukaryotic mRNA

• A modified base called 7-methylguanosine linked to the 5' end of a primary transcript with a phosphate bridge
• This structure is known as a 5' cap

Poly(A) Tail on Eukaryotic mRNA

• Polyadenylation adds an A sequence tail to the 3' end of a mRNA
• The poly(A) tail is approximately 250 adenine nucleotides
• Plays a role in mRNA export to the cytoplasm of the cell

RNA Splicing

• Not all stretches of primary transcripts are translated to protein in eukaryotes
• Protein coding regions are called exons; noncoding regions are called introns
• Spliceosomes composed of RNA + proteins splice together exons and remove introns
• Alternative splicing leads to different protein products from the same gene

Protein Translation and Structure

• Proteins are composed of amino acids
• The amino acid sequence determines the protein structure and function
• Proteins are made and folded into a specific shape to perform their functions correctly.

Amino Acid Structure

• Amino acids consist of a central carbon atom, amino group, carboxyl group, and variable "R" group

Hydrophobic Amino Acids

• These amino acids tend to be found in the interior of proteins
• Examples include alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan

Hydrophilic Amino Acids

• These amino acids tend to be found on the exterior of proteins
• Polar, basic, acidic
• Examples include asparagine, glutamine, lysine, arginine, aspartic acid, glutamic acid, serine, threonine, histidine

Special Amino Acids

• Glycine: small, flexible
• Proline: creates kinks
• Cysteine: forms disulfide bonds

Peptide Bond Formation

• Amino acids are linked together by peptide bonds to form polypeptides
• Peptide bonds are formed between the carboxyl group of one amino acid and the amino group of the next

Protein Nomenclature

• Protein = polypeptide
• Amino acid = residue

3-Dimensional Protein Shape

• The function of a polypeptide is dependent on its 3-Dimensional shape
• The shape is determined by its amino acid sequence.

Primary Protein Structure

• The primary structure of a protein is its amino acid sequence.
• Example: Ala-Met-Ala-Met

Alpha Helix

• The amino acid chain is tightly coiled in a right-handed manner
• Stabilized by hydrogen bonds between different parts of the peptide backbone

Beta Sheet

• Adjacent polypeptide chains can run parallel or antiparallel
• Stabilized by hydrogen bonds between different parts of the peptide backbone