BSCI410 Exam 3 + Final Stuff

Transposable Elements

DNA that has evolved the ability to move from place to place within a genome

→ “Jumping genes”

Can be highly disruptive to genes and chromosomes, yet also drive evolution

Don’t jump all the time. Movement can be activated by stressors like heat shock, radiation, tissue culturing, pathogen infection, or cancer.

They have a major impact on shaping genome structure and function

Effects of Transposable Elements

Genetic instability and disease

→ TEs can cause deleterious mutations, disrupt gene functions, and contribute to cancers or genetic disorders

Gene Regulation

→ TEs often contain regulatory sequences (promoters, enhancers) that when inserted close to genes, can rewire gene regulatory networks

Chromosome rearrangements

→ High concentrations of TEs can lead to unequal crossing over, causing deletions or duplications

Genetic Variability and Evolution

→ Through TRANSPOSITION (movement of TEs in the genome), TEs increase genetic variability, which can help populations adapt to environmental changes

Epigenetic Modification

→ Organisms use silencing mechanisms (DNA Methylation, histone modifications) to repress TEs. But this can silence the expression of genes close to the transposon.

TEs Background

Existence of TEs was inferred from genetic studies in Maize (Corn mottling was caused by movements of a TE into and out of a pigment gene)

Unrecognized for many years because DNA was believed to have fixed positions on chromosomes

TEs were thought to be “selfish” DNA. Have now been found in all organisms.

12.5% of the Drosophila genome are TEs

90% in Maize

TE Abundance in Human Genome

44% of Human Genome

Made of Retrotransposons
-LINEs: 20%

-SINEs: 13%

-HERVs: 8%

and DNA Transposons: 3%

Only a few LINEs and SINEs in the human genome are able to move due to mutations in the sequences needed for transposition

The HERVs and DNA transposons in the human genome are immobile relics.

DNA Transposon Mechanism

Deletion and then goes into the target site

Retrotransposon Mechanism

Copies itself and then goes into target site

Can grow in quantity across the genome

3 Types of Retrotransposons

Long Interspersed Elements (LINEs)

Short Interspersed Elements (SINEs)

Human Endogenous Retroviruses (HERVs)

LINEs and HERVs have a gene encoding REVERSE TRANSCRIPTASE called pol that transcribes mRNA into single-stranded cDNA

HERVs are similar in structure to retroviruses (RNA tumor viruses) and are flanked by LONG TERMINAL REPEATS (LTRs)

Retrotransposons: Transposons that move via an RNA INTERMEDIATE

How LTR (Long Terminal Repeat) Retrotransposons Move

1. Transcription of retrotransposon pol

2. Synthesis of cDNA by REVERSE TRANSCRIPTASE

3. Staggered cut is made in the genomic target site

4. Retrotransposon cDNA inserts into the target site

5. Original copy in the genome remains while new copy inserts into the other genomic location

DNA Transposons

Most have INVERTED REPEATS (IRs)

→ 10-200bp long at each end

and a gene encoding TRANSPOSASE gene, which recognizes the IRs and cuts at the border between IR and genomic DNA

How DNA Transposons Move

1. Transposase cleaves the P-element (Inverted Repeats + Transposase gene in the middle) and excises it

2. Then adds it to a new location

3. Repair of the original gap using a sister chromatid or homologous chromosome occurs. The DNA Transposon can either remain or be removed from its original location depending on whether it exists on the homologous chromosome or sister chromatid.

Modified P Elements to Modify Flies

1. Insert the desired DNA between the P element Inverted Repeats of a transformation plasmid (no transposase gene here)

2. Then insert a helper plasmid that encodes for the transposase gene. Thus, transposase is formed and can move the first plasmid’s segment, but not itself since it lacks IRs of its own.

3. Inject both into syncytial embryos.

Defective TE Copies

Many TEs end up with deletions during the transposition process

→ Deletion of PROMOTER for retrotransposon transcription

→ Deletion of REVERSE TRANSCRIPTASE/TRANSPOSASE GENE

→ Deletion of INVERTED REPEATS needed for DNA Transposons

Creates disparity between Autonomous TEs and Nonautonomous TEs

Autonomous TEs

Non-deleted TEs that can transpose on their own

Nonautonomous TEs

Defective TEs that require the activity of the autonomous enzymes in order to move (i.e. the sequence of interest in the drosophila transformation plasmid, which needs help from transposase in another plasmid)

How TEs disrupt genes and alter genomes

TE can insert within the coding region of a gene, inactivating it

TE can insert near a gene and affect its expression pattern

Alleles associated with TEs can be unstable

TE-Associated Mutant Alleles

Can often be unstable

A non-autonomous TE (Ds in picture) can hop into gene C and disrupt function

AC is an autonomous TE that cleaves out Ds and restores function

TEs altering genomes

TEs can also trigger spontaneous chromosomal rearrangements by unequal crossing over between TEs

→ Misalignment of homologous chromosomes or sister chromatids during meiosis/mitosis

Transposition can also cause relocation of a gene in the genome

→ Can occur when two copies of a TE integrate in nearby locations on the same chromosome

→ Then the transposase recognizes the outermost IR sequences and moves the intervening sequences to a different location

Gene Duplication caused by TEs

RETROTRANSPOSONS can cause gene duplication

By mistakenly reverse transcribing cellular mRNAs and inserting them somewhere into the genome

OR by unequal recombination between repeated sequences due to misalignment (unequal crossing over) during meiosis

New Functions acquired by Duplicated Genes

Gene Families: Sets of related genes with slightly different functions; most likely arose from gene duplications

→ The two copies of a duplicated gene are related, but they can diverge in sequence and function

→ Some gene families in vertebrates have HUNDREDS of members thought to be driven by TEs

Alternative Splicing by Transposase Gene limits TE Movement

One splice form produces TRANSPOSASE

The other produces a transposition REPRESSOR

The repressor COMPETES with transposase for binding to the Inverted Repeats (IRs)

The Sequence Hypothesis

DNA, RNA, and Proteins are COLINEAR

→ They all run only in one direction. If we know the sequence of DNA, the RNA and Amino Acid sequence can be identified.

Different Types of RNA Polymerases

RNA Pol 1 is typically for Ribosomal RNA

RNA Pol 2 is for mRNAs, lnc(Long NonCoding)RNAs, and miRNAs

RNA Pol 3 is for tRNAs, U6 sn(Small Nuclear)RNA, and SS rRNA

Plants have two additional RNA pols (IV and V) which Synthesize siRNA

RNA Polymerases do not require a primer, unlike DNA Polymerases.

→ Can do “de novo” synthesis from a DNA template.

→ Adds new ribonucleotides to the 3’-OH of the growing RNA chain

3 Main Phases of Transcription

1. Initiation

When RNA polymerase enzyme gets loaded onto the core promoter

→ Involves GERMINAL TRANSCRIPTION FACTORS (GTFs)

2. Elongation

3. Termination

*Regulation can happen at all phases, but mostly happens at Initiation

Steps of Initiation

Activator Binding

→ Transcription factor proteins bind to gene-specific cis-regulatory regions (like enhancers)

Coactivator Recruitment

→ Activators (are TFs, and don’t work alone) and recruit intermediary factors known as CO-ACTIVATORS

Pre-initiation Complex (PIC) Assembly

→ General Transcription Factors (GTFs) and RNA Polymerase II assemble into the pre-initiation complex at the CORE PROMOTER

Core Promoter

Contains specific sequences known as PROMOTER ELEMENTS that bind General Transcription Factors (GTFs).

No universal element. For example, only 24% of human promoters have a TATA box.

Often have multiple elements cooperate to control a single gene.

At active genes, the core promoter is in a NUCLEOSOME-FREE REGION.

General Transcription Factors (GTFs)

Bind to the core promoter and load RNA polymerase.

TBP (TATA Box-binding Protein), a subunit of TFIID, binds the TATA box.

TFIIB binds the BRE element.

TFIIB and TFIID recruit the rest of the Pre-Initiation Complex (PIC)

→ Includes TFIIF, which loads RNA Pol II (TFIIF is a stabilizing factor for RNA Pol II)

→ Also includes TFIIH. The HELICASE subunit of TFIIH melts the promoter DNA and unwinds it to allow RNA Pol II to function.

Elongation can be regulated by RNA Pol II Pausing

Elongation factor P-TEFb phosphorylates the CTD of RNA Pol II to PROMOTE elongation. Releases the pausing of RNA Pol II, starts it again.

→ RNA Pol II pausing is used to load RNA Pol molecules on genes for RAPID INDUCTION.

→ Used for loading RNA Pol II before the RNA might be needed, thus allowing transcription to start quickly again once the mRNAs are actually needed.

DNA Looping

Can Mediate LONG-RANGE interactions between CORE PROMOTER and distal CIS-REGULATORY SITES

Mediator complex forms a bridge between TFs bound at enhancers, and the Pre-Initiation Complex (PIC) bound at the CORE PROMOTER.

→ Mediator has 30 subunits in animals.

This interaction occurs through looping of intermediate DNA.

Transcription Factors

Are DNA binding proteins that control the rate of RNA Synthesis

Are SEQUENCE SPECIFIC

Are often MODULAR Proteins:

→ DNA BINDING DOMAIN directly interacts with a specific DNA sequence

→ DNA binding domains contain highly conserved regions

→ TFs can be grouped into FAMILIES by the type of DNA binding domain they contain

→ EFFECTOR DOMAIN alters the rate or probability of transcription (ACTIVATOR domain or REPRESSOR domain)

→ Are less well conserved than DNA binding domains

*The more complex an organism is, the more TFs they tend to have

Helix-Loop-Helix Transcription Factor Example

The HOMEODOMAIN is a highly conserved DNA binding domain found across eukaryotes

→ Many major TFs that regulate development contain Homeodomains (like HOX proteins)

How TFs “read” DNA Sequences

TFs make SPECIFIC interactions w/DNA

→ Direct interactions between specific amino acids and bases in the MAJOR GROOVE

Major groove is larger and has more opportunities for interactions and bonds to occur

→ Different minor groove interactions are not unique between some nucleobase combinations, thus not good for reading DNA

TFs as Multimers

Some TFs bind DNA as dimers, trimers, tetramers, etc.

i.e. p53 is a tumor suppressor protein

→ Is the most commonly mutated gene in human cancers

→ and is a TETRAMERIC transcription factor

Chromatin

In the eukaryotic nucleus, DNA is packaged together with proteins to make CHROMATIN.

Packaging is dynamically controlled, and chromatin compaction is therefore not homogenous in the nucleus.

→ Regulated by specific enzymes.

→ CHROMATIN REMODELING ENZYMES and HISTONE MODIFYING ENZYMES

Nucleosome

A basic unit of chromatin, consisting of ~145bp of DNA wrapped around an octamer of histone proteins

Histones: Highly conserved BASIC (positively charged) proteins that bind to DNA

→ Core histones include H2A, H2B, H3, and H4

→ Made of 2x H2AH2B dimers, and 1x H3H4 tetramer

Flexible N-Terminal tails extend out of the core octamer

→ Are subject to EXTENSIVE PTM

Histone proteins block the initiation of transcription (if at the promoter), but NOT elongation.

Nucleosome Stacking

Is favored because of interactions between the negatively charged DNA and the positively charged N-Terminal tails of the Histones (Lysine-rich)

Histone Acetylation

Associated with Transcriptional Activity

Is controlled by HISTONE ACETYLTRANSFERASE (HAT) and HISTONE DEACETYLASE (HDAC) enzymes

Compaction occurs when the Positive Lysine residues on the N-Terminal tails of the Histones interact with the negatively-charged DNA

→ Addition of an acetyl group neutralizes the positive charge on lysine, uncompacts DNA

→ Thus opens up DNA and allows transcription to occur

Acetylation can recruit additional proteins

→ Some protein domains are “readers” of histone modifications. I.e. BROMODOMAINS (BD) which bind acetylated lysine residues

→ CBP is a TF and a HAT (therefore a writer) that also has a BD (a reader)

TFs and Histone Acetylation

CBP being both a reader and writer allows it to spread and maintain acetylation on chromatin beyond where the TF initially binds

However, CBP is a COACTIVATOR and has no sequence specificity. Thus, needs to be recruited by an ACTIVATOR, another TF called DORSAL (Drl)

Drl recruits CBP to drive transcription of genes.

Dorsal (Drl) can also act as a repressor

Alone, Drl acts as an activator.

When Drl binding occurs in conjunction with Dead Ringer (Dri) binding, the two proteins recruit GROUCHO, a transcriptional REPRESSOR protein

Thus, Drl can act as both an activator and a repressor TF.

TF Cooperativity

TFs can act cooperatively to regulate genes

→ Can assist the binding of additional TFs by remodeling the surrounding chromatin

PIONEER TFs are a special class of TF that can “open” Chromatin and pave the way for other TFs

Effector Domain receives signals to control TF Action

These signals can be HORMONES, which are perceived by NUCLEAR RECEPTOR proteins

→ Nuclear Receptor proteins are both hormone receptors AND TFs

i.e. Cortisol, which interacts with the Ligand Binding Domain of GR

→ GR gets freed and can then enter the nucleus

→ The GR DNA binding domain (DBD) interacts with cis-regulatory elements known as GLUCOCORTICOID RESPONSE ELEMENTS (GREs)

Chromatin Immunoprecipitation with sequencing (ChiP-Seq)

An affinity purification based method allowing us to map the genomic binding sites of chromatin proteins.

→ To find out where TFs bind in the genome

Protocol:

1) Cross-link/fix samples with Formaldehyde

2) Lyse cells and isolate Chromatin

3) Fragment Chromatin into smaller pieces (100-600bp) (using Sonication or Enzymatic Digestion)

4) Affinity purify target using an antibody (Immunoprecipitation)

5) Isolate and sequence DNA

ChiP-seq can also be used to map the genomic locations of any chromatin-bound protein like Histone modifications. You just need an antibody.

ChiP-Seq revealed that TFs can have hundreds-thousands of binding sites in the genome

→ Identifying DNA patterns of TF binding motifs is called MOTIF ANALYSIS

Cell Differentiation

TF cascades generate patterns of gene expression

TF proteins can act hierarchically. A MASTER/INITIATOR TF can activate or repress downstream TFs.

The assortment of TF proteins in a cell determines the expression state of genes, and therefore cell structure and function.

Positive Feedback in Transcriptional Regulation

An initiator TF activates expression of a terminal selector TF

The terminal selector TF activates downstream differential genes, but ALSO binds to cis-regulatory cites upstream of ITS OWN gene.

This simple genetic circuit maintains a pattern of gene expression that PRESERVES CELL FATE

Gene Regulatory Networks

Small genetic circuits are combined to make Gene Regulatory Networks

A network of regulatory actions make a Program of Gene Expression

RNA Processing

The nascent pre-mRNA must be processed before it’s ready for translation

→ Occurs CO-TRANSCRIPTIONALLY (occurs simultaneously with transcription)

→ ONLY mature mRNA is exported to the cytoplasm for translation

The coding sequence in Eukaryotes is interrupted by non-coding INTRONS. RNA splicing removes introns from the message.

→ Whereas in bacteria it’s typically ready for translation immediately

The now-MATURE mRNA contains an uninterrupted coding sequence sandwiched between two non-coding UnTranslated Regions (UTRs).

→ The UTR’s function is in translation, mRNA localization, and mRNA Stability (like mRNA metadata)

3 Key Steps of RNA Processing

1. 5’ Capping

2. RNA splicing

3. 3’ cleavage and poly-adenylation

RNA Pol II C-Terminal Domain (CTD)

The CTD is a platform for recruiting RNA processing enzymes

→ Consists of tandem repeats of the hepta-peptide YSPTSPS

→ The CTD is phosphorylated at different residues over the course of transcription. Called the CTD CYCLE

5’ Capping Enzyme

One enzyme recruited by the CTD is the 5’ capping enzyme

→ adds a 7-METHYLGUANOSINE (m7G) cap to the 5’ end of the pre-mRNA

The 5’ cap is crucial for:

mRNA Stability

mRNA Exporting

mRNA Translation

CAP BINDING PROTEINS interact with the 5’ cap

DECAPPING ENZYMES can remove the 5’ cap to trigger mRNA degradation

RNA Splicing Details

There are conserved sequences at the 5’ and 3’ ends of introns that are recognized as SPLICE SITES

→ By enzyme called the SPLICEOSOME

The 5’ end of the intron is cleaved and then reacts with the -OH of the BRANCHPOINT ADENINE (occurs within the intron itself)

The free 5’ splice site is then joined with the 3’ splice site to LINK the two exons.

The circularized intron is released as an INTRON LARIAT

→ Can be degraded by the cell

Spliceosome

Catalyzes RNA Splicing (removal of introns)

Has a core of 5 Small Nuclear RiboNucleoProteins (snRNPs)

Each snRNP is a complex of proteins and a small nuclear RNA (snRNA)

→ snRNAs form the catalytic core of the spliceosome.

*The spliceosome is an example of a RIBOZYME (an RNA enzyme)