Regulation of Gene Expression
Regulation of Gene Expression: When, Where, and How Much?
This section delves into the intricate mechanisms that control gene expression, determining precisely when, where, and to what extent genes are activated or silenced.
Variable Gene Expression
Gene expression is not a static process; it varies significantly based on a multitude of conditions, reflecting the dynamic needs of an organism:
Time in life: Gene expression patterns change dramatically across different developmental stages, from embryogenesis through adulthood and aging.
Cell or tissue type: Specialized cells within an organism express distinct sets of genes, leading to their unique structures and functions (e.g., a neuron expresses different genes than a muscle cell).
Environmental conditions: External factors, such as nutrient availability, temperature, light, or the presence of toxins, can profoundly alter gene expression profiles.
Healthy vs. diseased states: Deviations in gene expression are often hallmarks of disease, providing insights into pathological processes and potential therapeutic targets.
Overview of Gene Expression Control Points
The regulation of gene expression occurs at multiple stages throughout the central dogma, influencing the flow of genetic information from DNA to functional proteins. The human genome contains approximately \approx 20,325 genes, which can give rise to about \approx 100,000 different mRNAs through processes like alternative splicing, and ultimately lead to roughly \approx 1 million distinct proteins.
Key points of regulation include:
Chromatin remodeling: Occurs in the nucleus, controlling the accessibility of DNA for transcription.
Transcription: The process of synthesizing RNA from a DNA template in the nucleus.
Alternate splicing: A post-transcriptional modification in the nucleus where primary RNA transcripts are processed in different ways to produce multiple messenger RNAs (mRNAs) from a single gene.
MicroRNAs (miRNAs) block protein synthesis: A post-transcriptional regulatory mechanism in the cytoplasm where small non-coding RNAs inhibit translation or promote degradation of target mRNAs.
Translation: The synthesis of protein from an mRNA template in the cytoplasm.
Protein folding: Essential for protein function; misfolding can lead to aggregation or degradation.
Post-translational modifications:
Polypeptides shortened: Proteolytic cleavage can activate or inactivate proteins.
Sugars added: Glycosylation is a common modification affecting protein structure, function, and trafficking.
Polypeptides aggregate: Can be a pathological outcome or a regulatory mechanism for protein stability and activity.
Chromatin Restructuring and Epigenetics
Chromatin refers to the complex of DNA and histone proteins found in eukaryotic cells. This intricate structure plays a crucial role in gene regulation:
Histone obstruction: When histone proteins are tightly bound to DNA, they can physically block the access of transcription machinery, thereby inhibiting gene expression. This blocking action is independent of the strength of the gene's promoter region.
Epigenetics: This term describes processes that alter gene activity without changing the underlying DNA sequence itself. Instead, epigenetic mechanisms modify chromatin structure to influence gene transcription.
Chromatin restructuring: This involves adjusting the degree of tightness with which histones bind to DNA. These adjustments are achieved through modifications to either the histone proteins themselves or the DNA directly.
Tighter chromatin (condensed state) generally correlates with little transcription because the DNA is less accessible.
Looser chromatin (relaxed state) generally correlates with greater transcription because the DNA is more accessible.
Detailed Epigenetic Mechanisms
DNA compaction is necessary to fit the long strands of chromosomes within the confines of the cell nucleus. However, this compaction must be reversibly undone for accurate gene transcription to occur.
Certain regions of chromosomes are constitutively compacted and rarely, if ever, relax for transcription, such as heterochromatin.
The field of epigenetics specifically investigates the patterns of DNA and associated proteins and how these patterns impact gene expression.
Key epigenetic modifications include:
Histone acetylation:
Mechanism: The addition of acetyl groups to positively charged histone proteins.
Effect: This modification neutralizes the positive charge of histones, which in turn decreases their affinity for the negatively charged DNA.
Result: The chromatin structure becomes looser, increasing the availability of DNA for transcription.
DNA methylation:
Mechanism: The addition of methyl groups (typically to cytosine bases in CpG dinucleotides) directly to the DNA itself.
Effect: DNA methylation generally increases the affinity of histones for DNA (often by attracting methyl-binding proteins that recruit histone deacetylases, leading to tighter chromatin).
Result: The chromatin structure becomes tighter, decreasing the availability of DNA for transcription.
Transcriptional Regulation: Induction and Repression
Transcriptional regulation primarily controls the initiation and elongation phases of RNA synthesis:
Repression:
Mechanism: A specific repressor protein binds to an operator region located typically downstream of the promoter region.
Effect: This binding physically or allosterically blocks the progression of RNA Polymerase (RNA Pol), thereby blocking the elongation of transcription of the gene.
Induction:
Mechanism: An inducer molecule binds near the promoter region of a gene.
Effect: This binding facilitates the recruitment or activity of RNA Polymerase (RNA Pol), thereby increasing the rate of initiation of transcription.
Comprehensive Summary of Gene Expression Regulation Mechanisms
Regulatory mechanisms can either increase or decrease the overall expression of a gene, from DNA to protein:
Mechanisms that Increase Gene Expression:
Relaxed chromatin: Epigenetic modifications (e.g., histone acetylation) lead to a more open chromatin structure.
Induction of transcription: Presence of inducer molecules or activators promoting RNA synthesis.
Stable mRNA: mRNA molecules with longer half-lives allow for more protein synthesis before degradation.
Strong ribosomal binding sequence on mRNA: Efficient binding of ribosomes to mRNA leads to higher rates of translation initiation.
Strong splice sites on primary transcript: Accurate and efficient removal of introns, leading to functional mature mRNA.
Long-lived protein: Proteins that are stable and resistant to degradation remain functional for longer periods.
Mechanisms that Decrease Gene Expression:
Tight chromatin: Epigenetic modifications (e.g., DNA methylation, histone deacetylation) lead to a condensed chromatin structure.
Repression of transcription: Binding of repressor proteins or lack of activators inhibits RNA synthesis.
Short-lived mRNA: mRNA molecules with shorter half-lives (e.g., targets of RNA interference - RNAi) are rapidly degraded, limiting protein synthesis.
Weak ribosomal binding sequence on mRNA: Inefficient ribosomal binding leads to lower rates of translation.
Weak splice sites on primary transcript: Poorly recognized splice sites can lead to inefficient or incorrect splicing, producing non-functional mRNA.
Short-lived protein: Proteins that are rapidly degraded have a shorter functional lifespan.
Experimental Techniques for Gene Expression Analysis
mRNA Microarray
The mRNA microarray is a powerful tool to simultaneously measure the expression levels of thousands of genes under different conditions.
Preparation: Small amounts of DNA sequences, each corresponding to a specific gene, are meticulously dotted onto a solid surface, such as a glass slide or a silicone chip.
Sample processing: mRNA is extracted from a biological sample (e.g., cells or tissue) that has been subjected to particular experimental conditions.
Probe generation: The isolated mRNA is then used as a template to synthesize fluorescently labeled complementary DNA (cDNA) probes. The intensity of the fluorescence reflects the relative abundance of each mRNA molecule in the original sample.
Hybridization and detection: These fluorescent probes are then hybridized to the complementary DNA spots on the microarray. The binding of the probes to their target genes is detected by scanning the chips for fluorescence.
Interpretation: Differences in transcription levels are visualized by color patterns:
High levels of transcription for a particular gene are typically reflected by a green dot on that gene spot.
Low levels of transcription for a particular gene are typically reflected by a red dot on that gene spot.
Analysis: Specialized computer software is used to analyze the complex patterns generated, facilitating the comparison of gene expression profiles across multiple samples to identify differential expression.
2D Gel Electrophoresis of Proteins
Two-dimensional (2D) gel electrophoresis is an analytical technique used to separate and visualize a wide range of proteins from a biological sample.
Protein purification: All proteins present in a given sample are first extracted and purified.
Two-dimensional separation: The proteins are separated based on two distinct physical properties in sequential steps:
First dimension (Charge): Proteins are separated according to their isoelectric point (pI), which is the pH at which a protein carries no net electrical charge. This is typically achieved using isoelectric focusing (IEF).
Second dimension (Mass): Following separation by charge, proteins are further separated by their molecular mass using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Visualization and analysis: The result is a gel with a unique pattern of protein spots, where each spot ideally represents a single protein species. Computer software is then employed to compare these complex protein patterns obtained from different samples, allowing for the identification of proteins that are differentially expressed or modified between conditions.