CELL BIOLOGY - CLUTCH CH. 7 - GENE...

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CELL BIOLOGY - CLUTCH

CH. 7 - GENE EXPRESSION

CONCEPT: CONTROL OF GENE EXPRESSION BASICS

● Gene expression is the process through which cells selectively ______________ to express some genes and not others □ Every cell in an organism is a clone because they all contain an identical copy of DNA

- Different appearances and function depends on selectively expressing certain genes and not others

□ Gene expression controls the expression of proteins and RNAs

□ Cell differentiation is the process by which a cell becomes _______________________ for a particular function

- Differentiation is entirely directed by gene expression control

- Allows for the development of multicellular organisms with diverse cell types

EXAMPLE: Differentiation of a stem cell into many different blood cell types

● Gene expression can be ____________________________ at various steps in the DNA to RNA to Protein pathways □ Gene expression can be controlled by external signals

- One example is through hormones

□ Housekeeping genes are genes expressed in every cell because they are critical for cellular life

- Examples include: Ribosomal genes, RNA polymerase genes, DNA repair genes



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EXAMPLE: The many ways gene expression can be controlled

PRACTICE:

1. Gene expression is defined as which of the following? a. Genes are expressed because each cell contains a different set of genes b. Choosing which genes are expression by regulating only transcription c. Choosing which genes are expression because on only internal signals d. Cells choosing to express some genes and not others at many steps in the DNA to protein pathway



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2. True or False: Every gene is regulated differently in each cell type. a. True b. False



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CONCEPT: EPIGENETIC REGULATION OF GENE EXPRESSION

Epigenetic modifications

● Epigenetic modifications include heritable changes to the chromatin structure which alters gene expression.

- Including: histone modification, DNA methylation, DNA acetylation

- Histone Modification:

□ Histone methylation is the addition of a methyl group to certain amino acids on the histone protein

- Represses gene expression by stimulating chromatin condensation

- Catalyzed by histone methyltransferase (HMT) and removed by histone demethylase

□ Histone acetylation is the addition of an acetyl group to certain amino acids on the histone proteins

- Found in transcriptionally active chromatin because it stimulates an open chromatin structure

- Catalyzed by histone acetyltransferase (HAT) and removed by histone deacetylase (HDAC)

□ The histone code is the combination of methylation and acetylation events that alter chromatin structure and

regulate gene expression

- Recruit other regulatory proteins to the gene

- CpG (CG) islands are cytosine and guanine nucleotides that can be methylated or unmethylated

- Commonly found in promoter regions ~ high CG content ~1000-2000 nucleotides long

- Methylated CpG island= silenced gene

- Unmethylated CpG island= expressed gene

EXAMPLE: CpG islands

● Certain proteins can act as genetic activators

Non-methylated CpG islands= yellow



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or repressors

□ Gene activator or repressor proteins can modify local chromatin structure to change gene expression

- Nucleosome remodeling factors (NURF) are proteins that alter the arrangement of nucleosomes

- Do not effect methylation or acetylation

- Act by moving the histone protein octamer to a different DNA location

- Transcription Elongation factors: enzymes that remodel nucleosomes for transcription

- These proteins typically reside on the RNA polymerase tail, so that they can act during transcription

□ Activator proteins work synergistically

- Transcriptional synergy is when several activator proteins increase the rate of transcription

- Occurs when the new rate is higher than the rate sums of each activator working alone

EXAMPLE: Activated and condensed chromatin

Epigenetic Heredity

● Cells terminally differentiate, meaning that after differentiation, the daughter cells remain that cell type

□ Cell memory is the property that allows cells to pass patterns of gene expression to their daughter cells

- This is heredity that doesn’t include the DNA sequence – but instead the chromatin modifications

Luong, P. Basic Principles of Genetics (2009)



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□ Epigenetic inheritance is the property that allows organisms to pass patterns of gene expression to offspring

- This is heredity that doesn’t include the DNA sequence – but instead the chromatin modifications

- Genomic imprinting is when one parental gene copy remains active, while the other remains inactive

- Inactive copies remain methylated depending on source (sperm or egg)

- Two identical DNA sequences, but different chromatin modifications which effect expression

EXAMPLE: Altered methylation status of one gene (Avy gene) causes different phenotypes in genetically identical mice

● Chromosome wide chromatin structures can also be inherited by cellular offspring

□ X-inactivation is the transcriptional inactivation of an entire X chromosome

□ X-inactivation initiation is random, meaning that both X copies have the same chance of being inactivated

- Once one has been chosen it remains inactive for all cellular division

□ X-inactivation initiation occurs after a few several thousand cells have formed

- Therefore a mosaic phenotype appears when these cells each choose different X chromosomes

- The alleles on each copy encode for a different appearance, which can be seen throughout the body

EXAMPLE: Calico cats are the result of X-inactivation



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CONCEPT: TRANSCRIPTIONAL REGULATORS OF GENE EXPRESSION

How Transcriptional Regulators Work

● Transcriptional regulators ____________________ gene expression by activating or repressing the transcription of genes □ Transcriptional repressors turn genes off and therefore inhibit transcription

- Can compete with activators for binding

- Can inhibit transcription via protein-protein interactions

□ Transcriptional activators turn genes on, and therefore activate transcription

- Help make promoters fully functional by connecting with RNA polymerase

□ Can work with coactivators or corepressors which help to control transcription

- Modifying chromatin structure

- Activating the regulatory protein

□ Mediator is a 24 subunit complex that acts as a connector between regulatory proteins and RNA polymerase

EXAMPLE: Comparison of activators and repressors

□ Rarely do they work alone, and require other interactions and _____________________________ to be fully functional

- Other transcription factors are recruited to regulate gene expression

- General transcription factors: bind to core promoter site (Ex: TFIIB, TFIIH)

- Sequence specific factors bind to regulatory sites to activate/repress expression

- Each gene is regulated differently

Activator

RepressorGene

Gene



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EXAMPLE: Combination of transcription factors results in gene regulation

DNA binding motifs

● There are four common DNA binding __________________________ that transcriptional regulators contain □ Helix-turn-Helix: One helix makes contact with the DNA, while the other helix stabilizes the interaction

- Homeodomains are found on Hox genes which are crucial for proper development

□ Zinc Finger: Has repeats of cysteine and histidine that bind zinc and fold into a finger-like structure to bind DNA

□ Leucine Zipper: Dimerization of alpha helices with many leucine residues can bind DNA

□ Helix-loop-Helix: Two alpha helices connected by a loop can bind DNA

□ Transcriptional regulators bind to regulatory DNA sequences between 10 and 10,000 nucleotides in length

- Regulator proteins are degenerate meaning they don’t need an exact sequence to bind

- They don’t necessarily bind to the DNA nucleotides – can recognize and noncovalently bind to the helix

EXAMPLE: DNA binding domains

Helix-Turn-Helix Zinc Finger

Helix-Loop-HelixLeucine Zipper



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● Prokaryotes use _____________________________________ RNA polymerase subunits to control gene transcription □ Sigma subunit of RNA polymerase is required to recognize a promoter

- Many sigma subunits exist – and each recognizes a different set of promoters

□ Gene expression is controlled by replacing the sigma subunits of RNA polymerase

EXAMPLE: Sigma factor replacement allows for gene regulation in prokaryotic cells

Types of Transcriptional Regulators

● Transcriptional regulators can ____________________ to sequences located near or far from the gene they’re regulating □ Promoter-proximal elements lie near to the promoter site

- Promoter binds RNA polymerase and orients it correctly so it can transcribe the gene

- Contains the initiation site where RNA synthesis begins

EXAMPLE: A promoter (yellow) recruiting RNA polymerase (green) to the gene (blue)

RNA polymerase

Three different sigma factors



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□ There are numerous regulators that bind to DNA sequences _____________________ from the gene

- Enhancer is a DNA site to which gene activators bind

- Can be upstream or downstream from gene, and usually 1000s nucleotides away from promoter

- DNA between enhancer and promoter loop out to allow the two regions to interact

- Silencers is a DNA site to which gene repressors bind. Acts similarly to an enhancer

□ Insulators (barrier elements) divide chromosomes into independent segments

- Prevents distant elements (enhancers) from acting on promoters in a different segment

□ Gene control region: entire DNA sequence involved in regulating and initiating transcription of a gene

EXAMPLE: Example of enhancer activation

Tryptophan Repressor and Lac Operon

● The amino acid _________________________________ is a major regulator of gene expression in prokaryotes □ Can bind to operons (stretches of many related genes) and inhibit transcription

- Tryptophan binds to a transcriptional repressor to activate it

- The activated repressors binds to regulatory sequences to inhibit genes involved in tryptophan creation



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□ Allows gene expression to be controlled by environmental levels of tryptophan

EXAMPLE: Control of the trp operon

● The lac operon controls the _______________________________ of lactose in E. coli □ No lactose available: The lac repressor binds and halts transcription of lac operon

□ Glucose available: the activator Catobolie activator protein (CAP) remains inactive, but no direct repression

□ Lactose available: the activator Catobolie activator protein (CAP) binds upstream of promoter and activates

EXAMPLE: Control of the lac operon



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PRACTICE

1. Which of the following is not a DNA binding motif? a. Zinc Finger b. Leucine Zipper c. Helix-loop-Helix d. Helix-zipper-Helix

2. What is the purpose of a transcriptional mediator? a. To mediate regulation between transcription and translation b. To mediate the process of transcription c. To mediate between regulatory proteins and RNA polymerase d. To mediate between RNA polymerase and DNA



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3. True or False: Enhancers can reside downstream of the gene they regulate. a. True b. False

4. If lactose is present, what happens to the lac operon? a. It is activated b. It is repressed



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5.



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CONCEPT: ACTION OF TRANSCRIPTION REGULATORS

Combinatorial Control

● Combination of regulator proteins _________________________ gene expression □ Multiple proteins work together to determine the expression of a single gene

- Usually the first protein has high affinity, and then binding increases the affinity of other proteins

- Limits the number of transcription regulators needed

□ Expression can be decided by a single regulator protein

- Works like an on/off switch by completing the combination

□ Combinations can control the generation of different cell types

- A few transcription regulators control sets of genes resulting in cell differentiation

□ Combinations can be controlled by environmental signals

- Response elements are DNA sequence in a promoter that can bind to regulator proteins

- Ex: Heat-shock response elements, hormones

EXAMPLE: Combinations of regulatory proteins control gene expression

Steps to Gene Activation

● Gene activation occurs in 6 steps 1. Regulatory proteins bind to an enhancer 2. This binding stimulate the DNA to form a loop which connects the enhancer and promoter 3. Activators interact with coactivators to alter chromatin structure 4. Activators interact with the mediator

Promoter

Promoter

GeneRNA pol.



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5. Mediator facilitates the correct positions of RNA polymerase 6. RNA polymerase begins transcribing EXAMPLE: Steps to gene activation

Nuclear Receptors and Hormones

● Nuclear receptors are transcriptional regulators that sense hormone (steroids) and regulate gene expression □ Nuclear receptors contain a few important _________________________

- N-terminal act as an activation domain

- Has a DNA binding domain

□ Inverted repeats are sequences of nucleotides followed by a reverse complement downstream

- Hormone response elements are inverted repeats that many nuclear receptors bind

Enhancer Promoter Gene

Enhancer Promoter Gene

1.

Promoter

Gene

2.

Promoter

GeneRNA pol.

3

4

5 6.



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EXAMPLE: Hormone activation of a gene

PRACTICE

1. Choose all of the following factors involved in combinatorial control of gene expression. a. Regulatory proteins b. Response elements c. Histone Proteins d. RNA polymerase e. Glycosylation



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2. What are nuclear receptors? a. Hormones b. Receptors found on the surface of the nucleus c. Receptors found on the surface of the cell d. Proteins in the nucleus that bind to hormones



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CONCEPT: POST TRANSCRIPTIONAL REGULATION

RNA Processing, Translation, and Degradation

● Regulation of mRNA after transcription is a major way to control gene expression

□ RNA processing includes alternative splicing, preparation for nuclear export, and RNA editing

- Improperly processed mRNAs are not exported and translated

□ RNA translation can be controlled

- Phosphorylation of eIFs (bind to 3’ mRNA to promote translation) can globally inhibit cellular translation

- Phosphorylated eIF cannot exchange GDP for GTP and therefore can’t promote translation

- Translational repressors are proteins that control translation of specific mRNAs

□ mRNA degradation rates vary and are one way to regulate gene expression

- Shorter poly(A) tails are less stable than longer tails

- Exosomes degrade mRNA from 3’ to 5’ via exonucleases

- P bodies are nuclear mRNA processing bodies that degrade mRNA from 5’ to 3’

- Nonsense mediated decay degrades improperly spliced mRNA that lack proper protein coding regions

- When stop codon is in wrong place

EXAMPLE: RNA processing, translation, and degradation can control gene expression



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RNA Interference

● Various types of regulatory RNAs can control gene expression

□ Small interfering RNA (siRNA) is one form of RNA mediated inhibition evolved to protect cells from viruses

1. siRNAs are double stranded RNA that enter cells via foreign objects

2. The enzyme dicer cleaves siRNAs into small fragments

3. The RNA induced silencing complex (RISC) binds these fragments and degrades one strand

4. The single stranded siRNA can bind to a complementary mRNA – which is then degraded by RISC

- Argonaute is the catalytic component of RISC that cleaves the mRNA

EXAMPLE: siRNA mediated mRNA degradation



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□ Micro RNA (miRNA) is a second form of RNA mediated inhibition that is encoded by the genome

1. miRNAs are single stranded RNAs created through transcription (~22 nucleotides long)

2. After transcription, miRNAs form hairpins or loops based on complementary RNA sequences

3. DROSHA cleaves the loops and the free miRNA associates with RISC

4. The processed miRNA binds to a 3’ UTR end of mRNA and inhibits expression via RISC degradation

- Each miRNA can regulated ~200 mRNAs

EXAMPLE: miRNA processing and complex formation with RISC



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□ Other noncoding RNAs regulate gene expression

- Piwi-interacting RNA (piRNAs) suppress movement of transposons

- Long noncoding RNAs are 200+ nucleotides in length and regulate gene expression

Protein Regulation

● Regulation of mRNA after transcription is a major method of controlling gene expression

□ Protein modifications can inhibit or activate protein _______________________

- Examples include: Protein phosphorylation, dephosphorylation and cleavage

□ Protein degradation controls a proteins function

- Ubiquitin labeling - proteasome destruction and lysosomal destruction

- Degrons are protein regions that control a protein’s destruction

EXAMPLE: Presence of degron reduces protein presence over time



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PRACTICE

1. Choose all of the following post-transcriptional regulators of gene expression.

a. Micro RNAs

b. siRNAs

c. RNA Polymerase Degradation

d. Exosomes

2. True or False: When the siRNA interacts with RISC for the first time it is single stranded.

a. True

b. False



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3. What is the name of the enzyme that cleaves the miRNA in the nucleus before it travels to the cytoplasm to exert its

effects?

a. RISC

b. Argonaut

c. DROSHA

d. RNA Polymerase

4. What is the name of the region on a protein that controls its degradation over time?

a. Degradation sequences

b. Ubiquitin

c. Ubiquitin binding site

d. Degron



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5. True or False: All non-coding RNAs are responsible for regulating gene expression.

a. True

b. False



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