Module 3.4: Turning Off The Signal

At What Points of Synthesis of Functional Protein can Gene Expression be Regulated?

1. Transcription initiation

2. RNA processing

3. Overall stability of RNA polymerase

4. Protein Synthesis

5. Protein Modification and Transport

6. Protein Degradation

In Situ Analysis:

• If we suspect a specific gene plays an important role in the embryonic development of the fruit fly, Drosophila, It is possible to identify which embryonic cells or tissues express the gene by analyzing where the corresponding mRNA can be found during development in the intact organism and this is known as in situ analysis

• The mRNA can be detected using a complementary probe with a fluorescent tag attached to it.

In Situ Hybridization:

• The complementary probe to detect mRNA would be a fluorescently labelled, short, single-stranded segment of DNA or RNA.

• The probe binds, or hybridizes, to the target mRNA molecule through complementary base pairing in a process called in situ hybridization.

• can be used to study the expression of one or a few genes of interest and gene expression over time as one develops and gets older.

DNA Microarray Techniques:

• These techniques developed in the mid-1990s and made it possible to examine thousands of genes at once, not just one or a few via in situ hybridization.

• They are largely based on complementary base pairing between nucleic acid strands and they are essentially an in vitro adaptation of the in situ hybrid technique: glass slides called DNA/gene chips containing tiny spots, each with a known DNA sequence representing a gene.

• Detect gene expression and investigate transcriptome.

DNA Microarrays:

• DNA microarrays can contain up to 100,000 oligonucleotides, or short fragments of nucleic acids, with each representing a different gene.

• can identify differences in gene expression between normal and cancerous cells.

• useful for visualizing gene expression across various cells, during different stages of development, and in response to different signals.

Transcriptome:

• the set of mRNA transcripts expressed by genes in a cell or organism.

Preparing Fluorescently labelled complementary DNA (cDNA) for Microarray Analysis (MAA):

• mRNA is isolated from cells.

• It can now be used as a template to create cDNA

• Now, an enzyme called reverse transcriptase can be used to make cDNA via reverse transcription from RNA to DNA.

• During this transcription, fluorescently labelled nucleotides are incorporated into the new cDNA.

• different cDNAs from different types of cells are labelled with different coloured fluorescent dyes so that they may be distinguished during MAA.

DNA Microarray process:

• Microarray chip has large number of oligonucleotide, representing different genes, fixed to a glass slide in a tightly spaced array.

• the DNA that’s fixed to these chips is of genes that are known to be present in a given cell, keeping in mind also that gene expression can very considerably at any given time.

Tested the Various Cells in Breast Cancer Example:

Each cell was fluorescently labelled:

• Normal breast epithelial cells: cDNA fluoresces green

• Breast carcinoma cells: cDNA fluoresces red

These 2 sample are combined in equal amounts and tested for hybridization with the single-stranded DNA molecules in the microarray chip.

The difference and intensities of fluorescence are then measured via the the special scanner at the different spots on the microarray slide.

Interpreting Microarray Analysis Results:

• Key to interpretation: active gene = mRNA production = more labelled cDNA post the reverse transcription process = lets more cDNA to hybridize with the corresponding DNA on the microarray chip = stronger fluorescent signals.

• When equal mixtures of labelled cDNA from normal breast epithelial cells and breast carcinoma cells are applied to a microarray chip, the two samples compete to bind to complementary DNA fragments at each spot.

Interpreting fluorescent colours in MicroArray Analysis:

• Green Spot = gene is expressed more highly in normal epithelial cells and is downregulated in breast carcinoma cells.

• Red Spot = gene is expressed more highly in breast carcinoma cells and is upregulated compared with normal epithelial cells.

• Yellow Spot: gene is expressed at approximately equal levels in both cell types.

• No Fluorescence Detected = gene may be inactive or expressed below the detection limit.

Genes producing more mRNA = brighter fluorescent spots

RNA interference (RNAi) and Gene Silencing via microRNA by destroying target mRNA:

•  some genes can be transcribed into mRNA without that mRNA ever being translated into protein.

• can occur through the activation of RNA interference (RNAi) machinery by short, noncoding regulatory RNA molecules such as microRNA (miRNA)

• MicroRNAs are transcribed from DNA using RNA polymerase and their precursor transcripts often form hairpin loops because of complementary base pairing within the RNA molecule.

• These hairpin loops are then processed into smaller RNA fragments that can activate the RNA interference machinery.

• The processed microRNA becomes incorporated into an RNA-induced silencing complex (RISC).

• The microRNA within the RISC complex contains sequences complementary to specific target mRNA sequences that require regulation.

• The microRNA binds to the target mRNA, often through imperfect or non-exact base pairing

• The microRNA binds to the target mRNA, often through imperfect or non-exact base pairing. Once this occurs, proteins within the RISC complex can inhibit translation and, in many cases, promote mRNA destabilization or degradation.

RNA interference via Small Interfering RNA (siRNA) to cut off target mRNA and destabilize it:

• processed in a manner similar to microRNAs and also become associated with the RISC complex.

• typically bind to their mRNA targets through near-perfect or exact sequence complementarity.

• They associate with the RISC complex and direct the cleavage, or cutting, of the target mRNA.

• This destabilizes the target mRNA and further contributes to the post-transcriptional regulation of gene expression.

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Post-Translational Mechanisms to Control Gene Expression:

1. Cleavage

2. Disulphide Bond Formation

3. Acetylation

4. Phosphorylation

5. Methylation

Post-translational modifications allow cells to activate or inactivate specific proteins, however the length of time a protein functions in a cell can be limited by a process called selective degradation.

Selective Degradation:

• Breaks down damaged, short-lived and regulatory proteins.

Selective Degradation via Proteosomes:

• Proteasomes are large protein complexes found in cells that break peptide bonds and degrade unneeded or damaged proteins.

• This enables a cell to regulate the concentrations of specific proteins by breaking long polypeptides into smaller fragments

• these smaller fragments can then be further degraded into amino acids and reused in subsequent rounds of protein synthesis

The Ubiquitin–Proteasome Pathway:

• The cell must accurately identify which proteins should be degraded via the Proteasome.

• This is done through a tagging mechanism

• target proteins are tagged with small proteins called ubiquitin through an enzymatic cascade, which is very ATP-dependent.

• This results in a polyubiquitin chain that marks the target protein for degradation by the proteasome.

• The passage of the target protein through the proteasome is also largely ATP-dependent.

• ATP helps unfold the protein so it can be broken down into smaller fragments to degrade the protein.

The Uniquitin Tagging Process:

• The tagging process is largely ATP-dependent and involves three main steps:

1. Activation: Ubiquitin is activated by an enzyme.

2. Conjugation: Ubiquitin is transferred to the target substrate protein through the action of additional enzymes.

3. Ligation: Ubiquitin is attached to the substrate protein, and repeated cycles can build a polyubiquitin chain.