🧬Molecular Biology Unit 7 – Gene Regulation

Key Concepts and Terminology

• Gene regulation controls the expression of genes in response to internal and external stimuli
• Transcription factors are proteins that bind to specific DNA sequences to activate or repress gene expression
• Promoters are DNA sequences located upstream of a gene that initiate transcription and provide binding sites for RNA polymerase and transcription factors
• Enhancers are distant DNA sequences that can increase transcription of a gene by interacting with transcription factors and the promoter
• Silencers are DNA sequences that can decrease or silence gene expression by binding repressive transcription factors
• Chromatin structure refers to the packaging of DNA with histone proteins, which can influence gene accessibility and expression
• Epigenetic modifications, such as DNA methylation and histone modifications, can alter gene expression without changing the DNA sequence

DNA Structure and Gene Organization

• DNA is a double-stranded helix composed of nucleotides containing a phosphate group, a sugar (deoxyribose), and one of four nitrogenous bases (adenine, thymine, guanine, or cytosine)
• Genes are segments of DNA that encode functional products, such as proteins or RNA molecules
• Genes are organized into coding regions (exons) and non-coding regions (introns)
  • Exons contain the genetic information that is translated into proteins
  • Introns are removed during RNA splicing before translation occurs
• Regulatory sequences, such as promoters and enhancers, are located near genes and control their expression
• Chromatin is organized into nucleosomes, which consist of DNA wrapped around histone protein octamers
  • Nucleosomes can be further condensed into higher-order structures, such as chromatin fibers and chromosomes
• The organization of genes and regulatory elements can vary between prokaryotes and eukaryotes

Transcription Basics

• Transcription is the process of synthesizing RNA from a DNA template
• RNA polymerase is the enzyme responsible for catalyzing the synthesis of RNA during transcription
• Transcription initiation occurs when RNA polymerase binds to the promoter region and begins synthesizing RNA
  • In prokaryotes, the primary RNA polymerase is called RNA polymerase holoenzyme, which consists of a core enzyme and a sigma factor
  • In eukaryotes, there are three main types of RNA polymerase (I, II, and III), each responsible for transcribing different classes of genes
• Transcription elongation involves the progressive addition of nucleotides to the growing RNA chain by RNA polymerase
• Transcription termination occurs when RNA polymerase reaches a termination signal and releases the newly synthesized RNA molecule
• The resulting primary transcript undergoes post-transcriptional modifications, such as 5' capping, 3' polyadenylation, and splicing (in eukaryotes)

Prokaryotic Gene Regulation

• Prokaryotic gene regulation primarily occurs at the transcriptional level
• The lac operon is a classic example of prokaryotic gene regulation in E. coli
  • The lac operon consists of three genes (lacZ, lacY, and lacA) that are involved in lactose metabolism
  • The operon is regulated by the lac repressor, which binds to the operator sequence and prevents transcription in the absence of lactose
  • When lactose is present, it binds to the lac repressor, causing it to dissociate from the operator and allowing transcription to occur
• The trp operon is another example of prokaryotic gene regulation, which controls the synthesis of tryptophan
  • The trp operon is regulated by the trp repressor, which binds to the operator sequence in the presence of tryptophan, preventing transcription
• Attenuation is a mechanism of transcriptional regulation in prokaryotes that involves the formation of alternative RNA secondary structures, which can terminate transcription prematurely

Eukaryotic Gene Regulation

• Eukaryotic gene regulation is more complex than prokaryotic gene regulation and occurs at multiple levels
• Transcriptional regulation in eukaryotes involves the interaction of transcription factors with promoters and enhancers
  • General transcription factors (GTFs) are required for the assembly of the pre-initiation complex and the recruitment of RNA polymerase II to the promoter
  • Specific transcription factors bind to enhancers and regulate gene expression in response to various stimuli
• Chromatin structure plays a crucial role in eukaryotic gene regulation
  • Euchromatin is a loosely packed form of chromatin that is associated with active gene expression
  • Heterochromatin is a tightly packed form of chromatin that is associated with gene silencing
• Insulators are DNA sequences that can prevent the spread of heterochromatin and block the interaction between enhancers and promoters
• Alternative splicing is a post-transcriptional mechanism that allows for the production of multiple protein isoforms from a single gene

Epigenetic Mechanisms

• Epigenetic mechanisms are heritable changes in gene expression that do not involve alterations in the DNA sequence
• DNA methylation is the addition of methyl groups to cytosine residues, typically in CpG dinucleotides
  • DNA methylation is associated with gene silencing and is maintained by DNA methyltransferases (DNMTs)
  • DNA demethylation can occur passively during replication or actively through the action of enzymes such as TET proteins
• Histone modifications, such as acetylation, methylation, and phosphorylation, can alter chromatin structure and gene expression
  • Histone acetyltransferases (HATs) add acetyl groups to histones, which is associated with increased gene expression
  • Histone deacetylases (HDACs) remove acetyl groups from histones, which is associated with decreased gene expression
• Chromatin remodeling complexes, such as SWI/SNF, can alter the position or composition of nucleosomes, affecting gene accessibility
• Non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), can regulate gene expression through various mechanisms

Post-Transcriptional Regulation

• Post-transcriptional regulation occurs after transcription and can modulate gene expression at the level of RNA processing, stability, and translation
• RNA splicing is the process of removing introns and joining exons to form a mature mRNA
  • Alternative splicing can generate multiple mRNA isoforms from a single pre-mRNA, increasing protein diversity
• RNA editing is the modification of specific nucleotides in an RNA molecule, such as the conversion of adenosine to inosine (A-to-I editing)
• RNA stability can be regulated by elements in the 5' and 3' untranslated regions (UTRs) of mRNAs, such as AU-rich elements (AREs)
• Translational regulation can occur through the action of RNA-binding proteins (RBPs) and microRNAs (miRNAs)
  • RBPs can bind to specific sequences in mRNAs and influence their stability, localization, or translation
  • miRNAs are small non-coding RNAs that can bind to complementary sequences in mRNAs and promote their degradation or inhibit their translation

Practical Applications and Techniques

• Studying gene regulation has important applications in fields such as medicine, agriculture, and biotechnology
• Microarrays and RNA-sequencing (RNA-seq) are techniques used to measure gene expression levels on a genome-wide scale
• Chromatin immunoprecipitation (ChIP) is a method used to identify DNA-protein interactions, such as the binding of transcription factors to specific genomic regions
• CRISPR-Cas9 is a powerful gene editing tool that can be used to modify gene expression or correct genetic mutations
  • CRISPR-Cas9 consists of a guide RNA (gRNA) that directs the Cas9 endonuclease to a specific DNA sequence, where it creates a double-strand break
  • The double-strand break can be repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR), allowing for precise gene editing
• Reporter gene assays, such as luciferase assays, can be used to study the activity of promoters and enhancers
• Transgenic organisms, such as mice and plants, can be generated to study the effects of specific genes or regulatory elements in vivo