🧬Biochemistry Unit 10 – Regulation of Gene Expression

Key Concepts and Terminology

• Gene expression involves the process of transcribing DNA into RNA and translating RNA into proteins
• Regulation of gene expression controls when, where, and how much of a gene product is produced
• Transcription factors are proteins that bind to specific DNA sequences to regulate transcription
• Enhancers are regulatory sequences that increase transcription of a gene from a distance
• Silencers are regulatory sequences that decrease or prevent transcription of a gene
• Chromatin structure plays a role in regulating gene expression through modifications like histone acetylation and DNA methylation
• Post-transcriptional regulation includes mechanisms like RNA splicing, RNA stability, and RNA localization

DNA Structure and Gene Organization

• DNA is a double-stranded helix composed of nucleotides containing a phosphate group, a sugar (deoxyribose), and a nitrogenous base (A, T, C, or G)
• Genes are segments of DNA that encode functional products like proteins or RNA molecules
• Promoters are DNA sequences located upstream of a gene that initiate transcription and determine the transcription start site
• Exons are coding regions of a gene that are present in the mature mRNA after splicing
• Introns are non-coding regions of a gene that are removed from the pre-mRNA during splicing
• Untranslated regions (UTRs) are sections of the mRNA that are not translated into protein but play a role in mRNA stability and translation efficiency
• Chromatin structure influences gene expression through the packaging of DNA around histone proteins

Transcription Basics

• Transcription is the process of synthesizing RNA from a DNA template by RNA polymerase
• RNA polymerase binds to the promoter region and initiates transcription at the transcription start site
• The template strand of DNA is used to synthesize a complementary RNA strand in the 5' to 3' direction
• Transcription proceeds through three stages: initiation, elongation, and termination
• Transcription factors and regulatory elements influence the rate and specificity of transcription
• The resulting primary transcript (pre-mRNA) undergoes processing, including 5' capping, 3' polyadenylation, and splicing, to form mature mRNA
• Different types of RNA polymerases transcribe different classes of genes (RNA polymerase I, II, and III in eukaryotes)

Regulatory Elements and Sequences

• Promoters contain core elements like the TATA box and initiator sequence that are recognized by general transcription factors and RNA polymerase
• Enhancers are cis-acting regulatory sequences that increase transcription of a gene from a distance
  • Enhancers can be located upstream, downstream, or within introns of the gene they regulate
  • Enhancers function by binding transcription factors and recruiting coactivators to the promoter
• Silencers are cis-acting regulatory sequences that decrease or prevent transcription of a gene
  • Silencers can be located upstream, downstream, or within introns of the gene they regulate
  • Silencers function by binding repressor proteins that inhibit transcription
• Insulators are DNA sequences that prevent inappropriate interactions between neighboring genes and regulatory elements
• Response elements are specific sequences that bind transcription factors in response to cellular signals (hormones, stress, etc.)

Transcription Factors and Activators

• Transcription factors are proteins that bind to specific DNA sequences and regulate transcription
• General transcription factors (GTFs) are required for the assembly of the transcription initiation complex and basal transcription
• Activators are transcription factors that increase the rate of transcription by binding to enhancers or promoters
• Activators often contain DNA-binding domains that recognize specific sequences and activation domains that interact with coactivators and the transcription machinery
• Coactivators are proteins that bridge the interaction between activators and the transcription machinery and help modify chromatin structure
• Transcription factor binding sites are often clustered in regulatory regions, allowing for combinatorial control and fine-tuning of gene expression
• Signal transduction pathways can modulate the activity of transcription factors through post-translational modifications (phosphorylation, acetylation, etc.)

Repressors and Silencers

• Repressors are transcription factors that decrease or prevent transcription by binding to silencers or promoters
• Repressors can act through various mechanisms, such as competing with activators for binding sites, recruiting corepressors, or directly interfering with the transcription machinery
• Corepressors are proteins that interact with repressors and help mediate transcriptional repression
• Repressors often contain DNA-binding domains that recognize specific sequences and repression domains that interact with corepressors and the transcription machinery
• Some repressors can act as activators in different contexts, depending on the presence of specific cofactors or cellular signals
• Silencers can also function by inducing chromatin modifications that make the DNA less accessible to the transcription machinery

Epigenetic Regulation

• Epigenetic regulation involves heritable changes in gene expression without alterations to the DNA sequence
• DNA methylation is the addition of methyl groups to cytosine residues, typically in CpG dinucleotides, and is associated with gene silencing
• Histone modifications, such as acetylation, methylation, and phosphorylation, can alter chromatin structure and affect gene expression
  • Histone acetyltransferases (HATs) add acetyl groups to histones, making chromatin more accessible and promoting transcription
  • Histone deacetylases (HDACs) remove acetyl groups from histones, making chromatin less accessible and repressing transcription
• Chromatin remodeling complexes, such as SWI/SNF, can alter the positioning of nucleosomes and regulate access to DNA
• Noncoding RNAs, such as microRNAs and long noncoding RNAs, can also participate in epigenetic regulation by targeting specific mRNAs or recruiting chromatin-modifying complexes

Post-Transcriptional Regulation

• Post-transcriptional regulation involves mechanisms that control gene expression after transcription but before translation
• Alternative splicing allows for the production of multiple protein isoforms from a single gene by selective inclusion or exclusion of exons
• RNA editing involves the modification of specific nucleotides in the RNA sequence, such as the conversion of adenosine to inosine (A-to-I editing)
• RNA stability and degradation can be regulated by elements in the UTRs, such as AU-rich elements (AREs), which recruit RNA-binding proteins that promote or prevent degradation
• RNA localization involves the transport of mRNAs to specific subcellular compartments for localized translation, which is important for processes like embryonic development and synaptic plasticity
• Translational regulation can occur through mechanisms like ribosome binding, initiation factor availability, and upstream open reading frames (uORFs)
• RNA interference (RNAi) is a mechanism where small noncoding RNAs, such as microRNAs and siRNAs, target specific mRNAs for degradation or translational repression

Practical Applications and Techniques

• Reporter genes, such as luciferase or green fluorescent protein (GFP), can be used to study promoter and enhancer activity by placing them under the control of regulatory sequences
• Chromatin immunoprecipitation (ChIP) is a technique used to identify DNA sequences bound by specific proteins, such as transcription factors or histone modifications
• DNA footprinting is a method used to determine the binding sites of proteins on DNA by exploiting the protection of bound regions from enzymatic or chemical cleavage
• Electrophoretic mobility shift assay (EMSA) is a technique used to study protein-DNA interactions by observing the migration of protein-bound DNA fragments in a gel
• RNA-seq is a high-throughput sequencing method used to quantify and analyze the entire transcriptome, providing information on gene expression levels, alternative splicing, and noncoding RNAs
• CRISPR-Cas9 is a powerful genome editing tool that can be used to modify regulatory sequences, create gene knockouts, or introduce specific mutations for functional studies
• Transgenic organisms, such as mice or plants, can be generated to study the effects of specific gene regulatory elements or mutations in a living system