Applied Lectures #4-6: Theme 2 Module 3 - Theme 3 Module 4

Development of eukaryotic organisms depend on

Gene regulation and signal sequences

Gene regulation significance

Allows for the creation of various specialized cells derived from a single-celled zygote

Transcription factors

Proteins that bind to specific DNA sequences to control transcription. Work together with other proteins that cause changes in gene expression in successive cell divisions

Transcription factors significance

Determine the pathway that a specific cell will follow and determine the final mature/differentiated cell type

Eukaryotic vs Prokaryotic transcription

Similar: 

• RNA Pol

• Activating/repressing proteins

Different:

• Compartmentalization

• Each gene controlled by its own promoters and enhancers (E)

• Compact chromatin (E)

Chromatin remodeling

Unraveling of chromatin to initiate transcription

Why do we need chromatin remodeling

DNA in chromatin form is tightly wound, and thus genes are not expressed, need chromatin remodelling for transcription to occur

Chromatin structure

DNA wound around histones to form nucleosomes. 1 Nucleosome = 8 histones = 150 BP’s. DNA & histones bonded through + charged histone tails and - charged phosphates in DNA

Chromatin remodelling process

1. Transcription factor binds to enhancer site and recruits coactivator enzyme HAT

2. HAT attaches acetyl groups to lysine a.a’s along + charged histone tail to reduce + charge

3. Weakened + charge causes bond to weaken, allowing DNA to be unwound and transcribed

Chemical modifications

• Acetylation of lysine 

• Methylation of lysine & arginine

• Phosphorylation of serine & threonine

Alter charges of histone tails to weaken DNA bond and allow space for transcriptional proteins. # of groups correspond to different functions (Ex. 1 methyl = transcriptional activation, 3 methyl’s = repression)

Eukaryotic transcription factors

Bind based on structural and chemical complementarity. Have alpha-helical domains that bind to DNA through hydrogen bonds between a.a’s in alpha helix and nitrogenous bases in DNA

Types of eukaryotic transcription factors

Hair-loop-hair, helix-turn-helix, zinc finger, leucine zipper

Cis-sequences

Eukaryotic promoter regions required to initiate transcription

Core promoter

Binding site required for RNA Pol and transcription factors (Ex. TATA box, BRE region). Close proximity to initiation site

What is TATA box sequence recognized by

TATA-binding protein (TBP), subunit of the transcription factor TFIID

What is BRE region recognized by

TFIID general transcription factor

Enhancer sequences

Located upstream, bind specific transcription factors that interact with promoter to form transcriptional complex and enhance transcription of a gene. Distant from promoter but due to flexible nature of DNA via looping, far-away regions are brought closer. Adaptor/mediator proteins connect proteins from enhancer region to promoter region.

Transcriptional repressors

Bind to silencer regions positioned upstream of target gene to activate, causing the interference of general transcription factor assembly, and subsequent transcription

Blood cell progenitor differentiation

Progenitor cells activate transcription of globin proteins. 

1st half of hemoglobin: 2 alpha-globin

2nd half (fetus): 2 gamma-globin

2nd half (adult): 2 beta-globin

Blood cells & transcriptional regulation

While fetuses require gamma globin to bind oxygen better and garner enough oxygen in the womb, this is not required into adulthood. Thus, progenitor cells halt the transcription of gamma-globin and increases the transcription of beta-globin subunits as the fetus matures into adulthood

How is beta-globin transcription repressed in fetuses

In fetal blood cell progenitors, chromatin is tightly wound around the beta-globin gene to inhibit transcription, whereas the gamma-globin gene site is open to allow transcription

Chemical modification of cystine bases

Addition of methyl group to CpG islands (string of C and G) located near/in promoter regions represses transcription by altering shape of DNA binding site to prevent RNA Pol binding.

Epigenetic mechanism

Process influenced by external factors (ex. diet) that alters gene expression, causing functionally relevant and heritable changes to the genome (Ex. methylation is heritable, maintains repressed state)

How do transcriptionally repressed methylated promoter further repress transcription

Though RNA Pol can no longer bind, other proteins can ONLY bind to CpG methylated sequence, further inhibiting transcription. Ex. HDAC binding removes acetyl groups from nearby histones, allowing nucleosomes to reassemble to mask promoter/enhancer sequences

Prokaryotic vs Eukaryotic regulation

Prokaryotic:

• Genes are default “on” for transcription

Eukaryotes

• Genes are default “off” for transcription, must be remodelled to expose promoter sequence

When can genes be regulated

1. Transcription initiation

2. RNA processing

3. Protein synthesis

4. Protein modification & transport

5. Protein degradation

What is the benefit of multi-level regulation

A cell can rapidly alter the levels of active proteins in response to internal and external signals

How can examine patterns of gene expression

Analyze where the corresponding mRNA that is transcribed from that gene is found during development in situ.

in-situ hybridization

Detects site of mRNA transcription with fluorescently tagged complementary probe composed of RNA/DNA that binds/hybridizes to the target mRNA molecule. Limited by the number of genes it can examine. 

DNA microarraying

in-vitro, examines the expression of thousands of genes at once. Use glass slides containing tiny spots of known DNA sequences/genes. These probes detect gene expression

Benefits of DNA microarraying

Allow visualization of gene expression variation across different stages of development, cell types, and in response to cell signals. Identify differences in gene expression levels between normal and cancerous cells.

DNA microarraying & breast cancer

1. Grow healthy and cancerous cell cultures and isolate genes/mRNA transcribed by both cell types 

2. Isolated mRNA serves as template for synthesis of complementary DNA molecules using reverse transcriptase enzyme

3. Fluorescent nucleotides are synthesized as part of cDNA 

4. Combine equal amounts of mRNA from normal cells (green) and cancer cells (red) to microarray chip and measure fluorescence

5. GREEN = EXPRESSED MORE IN NORMAL CELLS

6. RED = EXPRESSED MORE IN CANCER CELLS

7. Intensity of light determines relative gene activity

mRNA degradation

mRNA must be degraded to ensure halting of gene expression. Ex. length of polyA tail regulates stability, thus must be degraded

Regulation at Translation

Short, non-coding regulatory double-stranded RNA molecules activate the RNA interfering machinery, which ensures that some mRNA are not translated. Ex. MicroRNA synthesized from RNA Pol forms hairpin loops due to complementary base pairing within itself that fragment to activate RNA machinery

RISC Complex

RNA-induced silencing complex, composed of microRNA that contain complementary sequences for mRNA sequences requiring regulation. Bind to mRNA in a non-specific manner to inhibit translation

siRNA’s

Exact complements of target mRNA sequence, bind to the complementary sequence via RISC complex and induce the cutting of target mRNA

Post-translational modifications

1. Cleavage

2. Disulphide bond formation

3. Acetylation

4. Phosphorylation

5. Methylation

Selective degradation

Dictate how long a protein functions in a cell. Proteasome’s help regulate the concentrations of specific proteins by breaking long polypeptides into small fragments of a.a’s that are further degraded and used in subsequent rounds of translation

Proteasomes

Large protein complexes that break peptide bonds and degrade unneeded/damaged proteins. 

How are proteins destined for degradation identified

Target proteins are tagged through enzymatic cascade with small ubiquitin proteins in an ATP-dependent process facilitated by ubiquitin activation, conjugation to target protein, and ligation. Multiple ubiquitin’s attached forms a polyubiquitin chain that attaches to tagged protein, allowing proteasomes to degrade.

tRNA structure

70-90 nucleotides long, hydrogen bonds between complementary bases allows for 4 double-helical segments and 3 loops. Can also fold on itself into an L shape. a.a attached to 3’ end’s CCA nucleotide sequence, terminal Adenine acts as actual attachment for a.a during tRNA molecule activation

tRNA anticodon region

Nucleotide triplet that forms complementary base-pairs with specific mRNA codon. Written 3’ to 5’ and align with mRNA codons in 5’ to 3’

Aminoacyl tRNA syntheases

Facilitates activation of tRNA molecule with specific a.a. Specific to each type of tRNA and corresponding a.a, 20 for each a.a. Active site recognizes the anticodon of tRNA and a.a attachment site, and catalyze covalent attachment through ATP hydrolysis, creating a charged tRNA molecule 

How many codons can mRNA code for

64

How many tRNA molecules are involved in translation

45, <60 so some can bind to more than one codon due to the wobble theory

Where does translation occur

Cytoplasm

Translation initiation in Eukaryotes

Translation initiation complex forms near the 5’ cap, scans sequence until AUG start codon found

Translation initiation in Prokaryotes

Due to lack of 5’ cap, translation initiation complex forms at Shine-Dalgarno ribosome binding sites, located few bases upstream of AUG

Polycistronic

mRNA codes for multiple proteins

What is beneficial about translation initiation in prokaryotes

It can occur along multiple regions of the sequence, allowing it to have specific open reading frames for more than one protein along a single mRNA strand (polycistronic). Works because prokaryotes have functionally related genes grouped together, and thus transcribed as a single unit from one promoter

Ribosomal initiation complex assembly 

1. Translation factors bind to 5’ cap, allowing for assembly of small ribosomal unit

2. Other initiation factors bind to charged tRNA 

3. Unit moves along mRNA in 5’ to 3’ direction until AUG is encountered

4. Allows for large ribosomal unit to bind to initiation complex through energy from GTP hydrolysis

Translation Elongation

As each subsequent tRNA enters and binds to A (aminoacyl) site, they each attach to a GTP-bound elongation factor which is hydrolyzed when the correct codon-anticodon pairing has been made. The deacylated tRNA move from P-site to E-site, and the subsequent tRNA enters the A-site, allowing for the release of the E-site tRNA

Peptidyl-transferase reaction

Facilitates formation of peptide bonds, catalyzed by the large ribosomal subunit 

Translation Termination

Detection of stop codon allows GTP-bound release factors to bind to the A-site and catalyze hydrolsis of bond between terminal amino acid and tRNA in P site, and causes the dissociation of ribosomal subunits 

What machinery is required in translation (prokaryotes and eukaryotes)

Large and small ribosomal subunits, mRNA molecule, and charged tRNA a.a’s, initiation, elongation, and termination factors

Beadle & Tatum Experiment Significance

one-gene-one-enzyme hypothesis established the relationship between genes and proteins using the idea that Neurospora can grow well on minimal medium because it has some enzymes that can convert simple substances into arginine, as Neurospora can synthesize its own arginine through metabolic pathways (Ornithine - Citrulline - Arginine)

Srb and Horowitz

Modified it to the one-gene-one-polypeptide hypothesis. Performed study on radiation treated Neurospora to determine whether there are specific genes that produced the enzymes in the metabolic cascade for the synthesis of arginine. To prevent radiation from causing mutations in the DNA Neurospora was raised on medium containing nothing apart from the 3 enzymes

Srb and Horowitz conclusion

Mutations in specific genes block individual steps in metabolic pathways, preventing the organism from making certain nutrients. By adding the missing intermediate or final product to the growth medium, they could rescue the mutants, demonstrating that each gene directs the production of a specific enzyme

Exceptions to the one-gene-one-polypeptide hypothesis

The human genome has 20-25,000 protein-coding genes, proving that more than one protein can be produced from one gene. Post-translational modifications of proteins allow for diverse proteins

Human proteome 

Full number of proteins expressed by genome

How are glucose levels regulated

Sensory responses in Beta islet cells of the pancreas lead control glucose levels. High levels = pancreas modulates synthesis and secretion of insulin to regulate

Insulin

Small effector protein produced by pancreatic beta cells that facilitates a response on target cells, thus reducing blood glucose levels

Where is glucose absorbed

In the mouth through thin epithelial surfaces in close contact to capillaries and blood vessels & in the microvilli of the small intestine also in close contact to capillaries and small blood vessels

Insulin biosynthesis

Regulated at the transcriptional and translational levels.

Insulin structure

P.p translated from gene is 110 a.a’s long, but the functional protein itself is made of 51 a.a’s.

Why does insulin decrease in length

Post-translational modifications cause decrease in length

Preproinsulin

Original a.a chain length as coded by the gene. Synthesized by bound ribosomes and transported to lumen where it is processed through cleavage of the signal sequence, creating a proinsulin molecule.

Proinsulin

Undergoes folding in ER along with the addition of 3 disulphide bonds after which it is cleaved in golgi to form an insulin dimer (A & B chains) and release a small C chain that previously linked the A & B chains

Protein folding

Occurs in the ER through assistance of chaperone proteins, then transported to golgi

Why does preproinsulin need post-translational modification

Allows the N and C-terminal a.a from the A and B chains to bind to the insulin receptors on the target cells

Why do we need post-translational modifications

Increase functional diversity of proteome

Types of post-translational modifications

Cleavage, folding & disulphide bridge formation, covalent attachment of other molecules, degradation

Types of covalent attachment modifications

Phosphorylation, Acetylation, Methylation (PAM)

Phosphorylation

Reversible post-translational modification, attachment of phosphate group to threonine, serine or tyrosine via kinases

Methylation

Attachment of methyl group

Acetylation

Attachment of acetyl group

Receptor Kinases

Type of monomeric receptor embedded in cell membrane that dimerizes when bound to signal, causing the activation of the cytoplasmic region of the receptor, which behave like kinase proteins to phosphorylate at many regions on the receptor tail to trigger a cellular response

Dimerization

2 Monomers pairing up

How is glucose absorbed into cells

Insulin signal activates receptor kinases, causing a series of cytoplasmic proteins to activate and lead to an intracellular response that activates the glucose transported proteins at the cell surface

Intracellular signal amplification

Amplifier proteins downstream from receptor activate, follow a positive-feedback loop for amplification. Double negative-feedback loops inhibit the inhibitor of a signal, providing fine control to extracellular responses

Where is glucose stored

Fat cells in adipose tissues convert to fatty acids and store as triglycerides. Liver and muscle cells store as glycogen

Alternative splicing of pre-mRNA’s

Allows pre-mRNA to be spliced at different junctions to create mature mRNA molecules that contain a different combination of exons

Isoforms

Formed during alternative splicing when some exons are removed like introns due to the spliceosome recognizing an exon as an intron. Can be from the same or different cell.

How does alternative splicing help in the regulation of gene expression

The same primary transcript can be spliced in different ways to produce mature mRNA isoforms that can produce different but related proteins, promoting genetic diversity

Alternative splicing example

Primary transcript of gene that codes for insulin receptor.

• Insulin receptor gene = 22 exons

• Exon 11 is removed from the mature mRNA in skeleton muscle cells and translated into a higher affinity version of the insulin receptor in muscle cells

• This allows them to have a higher glucose uptake in response to insulin signals

• Liver cells retain exon 11 leading to a lower affinity for glucose uptake

Liver cells vs Skeletal cell insulin receptor

Lower vs higher affinity.

Insulin feedback system

Negative, drop in blood glucose is detected, causing subsequent decrease in insulin secretion

What if insulin/insulin receptor was not processed correctly

Lack of ability to bind to insulin receptors. Insulin receptor error = cannot activate glucose transport proteins =  inability to take up glucose = hyperglycemia & diabetes

What do prokaryotes require for survival

Amino acids, vitamins, nucleotides, carbs, favoured temperatures

Housekeeping genes

Genes required 24/7 for normal functions, alway being transcribed and translated (Ex. structural proteins, RNA/DNA Pol)

Regulated genes

Can be turned on/off as needed

How do bacterial cells respond to changing environments

Alter the expression pattern of some regulated genes

E. Coli and altering gene expression patterns

• Glucose is preferred carb source, once used up bacterial growth is arrested

• Unique gene expression mechanism allows them to switch to an alternate fuel source 

• If E.coli is grown with glucose and lactose, the glucose will still be used up before lactose

• The products of glucose breakdown activate the switch to lactose use

How does E.Coli breakdown lactose

Using B-galactosidase to produce glucose and galactose. Enzyme is produced by transcription of the B-galactosidase gene, which only occurs when lactose is present and glucose is unavaliable

What does transcriptional modification target

Amount of mRNA produced, controls the binding of proteins to the promoter to inhbit/activate transcription

What does translational modification control

Translation of mRNA to proteins

What do post-translational modifications control

Modification and activation of proteins

What 2 factors do protein synthesis depend on

Rate of translation and mRNA stability (quickly degraded = less protein)

Which level of regulation is fastest

Post translational, enzyme-driven chemical changes that occur much faster than synthesizing new proteins, respond quickly to external stimuli. Allows cell to have a stock of inactive proteins that can later be activated

Which level of regulation is slowest

Transcriptional, needs cell to start from scratch, only used for drastic environmental changes (Ex. E.coli glucose depletion). MOST EFFICIENT, no energy/resource is wasted making a mRNA/pp unless it needs to