Lecture 9: Control of Gene Expression

Prokaryotic Reproduction

• Goal is reproductive efficiency, not fine-tuning.

• Makes sure their genetic code can be replicated as efficiently as possible.

Cells respond to changes in the environment…

• In eukaryotes, this regulates the roles of cells required at different stages of development, which is known as cell differentiation.

• E.g; stem cells can have multiple purposes depending on the needs for development.

Natural Selection

• Prioritized bacteria that only produces products necessary for survival and reproduction (think efficiency!)

• Regulation of enzymes can come in a few different forms:

  • Feedback inhibition…regulates activity! Helps cut off the first enzyme in this metabolic pathway.

  • Gene regulation…regulating production! Represented through an operon model.

• Operon models are NOT used by eukaryotes. Eukaryotes require splicing and fine-tuning. Prokaryotes can also express multiple different genes in a single transcript, while eukaryotes are mono…

Operons

• Defined as a cluster of functionally related genes under control of a single “on/off switch”

• The “switch” is known as an operator, which is a portion of the promoter that has a sequence that can be recognized by a compressor protein.

Repressible Operons

• Can be switched “on/off” by repressor proteins.

• These operons are usually active'; the repressor protein starts in an inactive form that cannot bind to the operator.

  • Requires a corepressor to bring it to an active state and repress the pathway by binding to the operator.

• The repressor protein being inactive means that this is a repressible operon.

• Anabolic; always building…ideal for prokaryotes!!

Inducible Operons

• The operon is normally off and requires an inducer to switch on.

• Why produce these enzymes if it is not being used? It would be a waste of energy!

• Difference between inducible and repressible operons comes down to energy efficiency.

• Catabolic; always breaking down…not as ideal!!

Positive Gene Regulation

• Operons can be subject to positive control…very different from positive feedback!

• Uses stimulatory proteins call activators.

• CAP (catabolic activator protein) is a prime example:

  • Glucose is the preferred source of E. coli, but if there is no glucose and only lactose, CAP will bind with cAMP (cyclical AMP) and the activated CAP will attract RNA Polymerase to promote transcription.

  • When glucose levels rise, CAP will release from cAMP.

Alternative Sigma Factors

• Sigma factors…without this, RNA Polymerase cannot recognize the promoter and “open up” the helix to begin transcription.

• Alternative sigma factors recognize different promoters…

NOTE: TTGACA is the -35 BOX and TATAA is the -10 BOX!

Transcriptional Regulation in Eukaryotes

• All organisms much regulate which genes are expressed when

• Gene expression is regulated through several stages

• All cells are genetically identical, so we require cell differentiation.

• The following are the steps for gene expression:

Step 1: Chromatin Structure & Remodeling

• Euchromatin…loosely packed chromatin (more favorable for detection by promoters!)

• Heterochromatin…tightly packed chromatin

• Changing tails of histones will change how densely chromatin packs together

• Modifications to histones and DNA are caused by epigenetic factors, which greatly effect gene expression represented by offspring.

• CANNOT BE DETECTED BY GENOTYPING OR GENOME SEQUENCING since it does not affect the physical genetic code of the DNA.

Histone Methylation: signal to densify and inhibit the transcription of chromatin

Histone Acetylation: signal to loosen and promote transcription of chromatin

DNA Methylation

• The addition of a methyl group to specific DNA bases to inhibit transcription

• In genomic imprinting methylation regulates expression of maternal or paternal alleles of certain genes.

Control Elements and Transcription Factors

• Control elements are noncoding portions of DNA that serve as binding site that regulate transcription.

  • Poly-A Signal Sequence…adds adenine (poly-A) tail to mRNA and signals end of transcription.

• Proximal Control Elements…located close to the promoter

• Enhancers…distant control elements which could be as far as being on another chromosome! Could also be in an intron, etc.

• Transcription factors fall into two categories…

  • General Transcription Factors…required for transcription; usually bind to the promoter to aid the recruitment of RNA polymerase

  • Regulatory Transcription Factors…NOT REQUIRED; they interact with specific control elements to enhance or inhibit transcription.

• Activator…type of transcription factor that “rides” along DNA backbone and binds to an enhancer to stimulate transcription of a gene.

  • Have two domains, one that binds to DNA and one that activates transcription

  • Facilitate protein-protein interactions required for gene expression

  • Can avoid problematic regions, hope to different chromosomes, or be knocked off by other proteins colliding.

• Repressor…inhibit expression of specific genes through multiple methods

  • Interferes with binding of DNA to general transcription factors or RNA polymerase (similar to competitive inhibition!)

  • Competes with activators to bind to the same regulatory sequence.

  • Active repressors…contains a functional domain that uses protein-protein interactions to inhibit transcription.

Coordinated Control

• We require the right combo of activators to express the right combo of proteins.

• REVIEW!! Don’t understand!

Nuclear Architecture and Gene Expression

• Chromosomes may congregate to dense areas of transcription factors

• How to test changes in gene expression:

  • Fluorescence in situ (FISH) uses fluorescent dyes attached to nucleic acid probes to identify the location of specific mRNA’s in intact organisms and tissue.

    • Not quantitative and limited to one section at a time

  • Real-time RT & PCR uses reverse transcriptase enzyme and mix it with the extracted RNA from your sample; uses cDNA as the template for PCR and the amount of fluorescent PCR products can be quantified to indicate upregulation or downregulation of expression.

mRNA Processing…pre-mRNA to mature mRNA

• Splice out introns and adding caps to the 5’ and 3’ ends of the mRNA.

  • caps ____ on the 5’ end and ___ on the 3’ end

  • caps protect from exonucleases that could break down the mRNA…acts as a buffer

  • hall-pass to get out of nucleus core

mRNA Degradation

• mRNA lifespan in the cytoplasm is key to determining protein synthesis

• mRNA could be intercepted!

• Untranslated regions (UTR could code for regions that could activate proteins or inhibit them, etc.

RNA Interference

• Significant amount of genome is noncoding “junk” despite it not actually being junk; it can still code for things other than proteins (promoters, enhancers, etc.)…noncoding RNA (ncRNA)

• RNA Interference (RNAi)…inhibition of gene expression via the actions of noncoding RNA molecules

  • occurs when noncoding RNAi combines with proteins to destroy mRNA before it reaches a ribosome.

• Can be known as MicroRNAs (miRNAs) and small interfering RNAs (siRNA’s) which are principal examples of interfering RNA molecules.

  • miRNAs are made by cells while siRNAs are made in labs

miRNAs

• Formation of “hairpin” structures are formed into single strands by the enzyme Dicer

• miRNAs associate with RISC (RNA Inducing Silencing Complex) which binds ot the complex and degrades the complementary mRNA

• key in gene regulation…removing the dicer enzyme is lethal to embryonic development since those hairpin structures never break down to single strands (e.g., limbs do not properly develop)

• miRNAs are found to regulate thousands of genes!

siRNAs (same function but different source)

• generates from exogenous (foreign) RNA or from endogenous RNA that form double stranded structures

• Likely evolved to defend against RNA viruses (specifically double-stranded RNA)

• Degrades complementary mRNA with greater specificity and different proteins compared to miRNA

• Delivered not just by a scientist, but by a virus

Initiation of Translation

• Initiating translation of specific mRNAs can be blocked by regulatory proteins binding to specific sequences and structure of the mRNA

• Can control how long the protein survives in a cell

• Proteasomes are giant protein complexes that bind to and degrade protein molecules

Cell Differentiation & Specialization

• starts at the zygote and ends at adult

• Cell differentiation/specialization…the process by which cells specialize in structure and function

• Morphogenesis…physical process that provides the organism its shape

• Egg does most of the work, Sperm just donates necessary DNA

• Cytoplasmic determinants are maternal substances in the egg that influence early development

• Egg’s cytoplasm contains RNA, proteins, and other unevenly distributed substances in the unfertilized egg.

• As the zygote divides by mitosis the cells contain different cytoplasmic determinants which leads to variation in gene expression.

• Induction…signal molecules from the embryonic cell’s environment cause transcriptional changes in nearby target cells; this results in cell specialization.

• Master control/regulatory genes are responsible for switching on required transcriptional factors (e.g., MyoD which produces proteins committed to becoming skeletal features)

• Morphogens are signaling molecules that influence the expression of transcription factors that effect morphogenesis…morphogen gradient (unequal distribution of morphogen) establishes an embryo’s axis of growth.

Gene Regulation & Cancer

• Cancer is caused in mutations in genes regulating cell cycle control

• Oncogenes…cancer-causing genes which are normal environmental genes that got fucked up

• Proto-oncogenes become oncogenes.

• Changes in…

  • list three things

• Tumor-suppressor genes

  • prevent uncontrolled cell growth

  • they do the following:

    • Repair damaged DNA

    • Control cell division

    • Inhibit cell cycle

    • Promote apoptosis (death of cells)