Transcriptional and post-transcriptional regulation in eukaryotes

Overview: what does eukaryotic chromatin structure affect?

The availability of DNA to transcription machinery (aka what is considered euchromatin and heterochromatin)

Overview: what do eukaryotes use to regulate gene expression (2)

Complex sets of protein-to-protein (regulatory proteins) and protein to DNA interactions

Overview: A eukaryote has multiple points for regulation. What are they? (4)

Transcriptional, post-transcriptional, translational, and post-translational

Describe the locations of transcription and translation in eukaryotes

Transcription occurs in the nucleus, and translation occurs in the cytoplasm

How is the RNA treated in eukaryotes? (aka what is done to it)

It gets processed

What are and what is the purpose of enhances and silencers?

They are cis-acting sequences that affect gene expression (along with promoter sequences).

They lead to differential expression of eukaryotic genes

Compare and contrast enhancer and silencer sequences (purpose/function and what they are bound by)

Enhancers increase the level of transcription while silencers repress transcription of certain.

Enhancers are bound by activator proteins, repressor proteins (opposite effect of enhancer), and their cofactors. Repressors are bound by repressor proteins

Describe the location of enhancers and silencers

They have variable distances (up to 1000 Kb) from their target genes + can be upstream, donwstream, or in introns

What are enhancers in yeast called

Upstream activator sequences (UAS)

What are some factors that make eukaryotic gene regulation so complex

1. each gene can have multiple regulatory sequences (prokaryotes usually have 2=promoter and operators)

2. the regulatory sequences can have multiple enhancers that control expression at different stages (see

3. the regulatory protein can act on many genes while

What do cofactors and expressors compete against

Activators and repressors

Do eukaryotes have operons

No (multiple promoters for many genes)

What are activator proteins?

Specific transcription factors that bind to enhancers and are responsible for higher levels of transcription/stimulating transcription

How do activator proteins increase transcription (broadly)

1. stimulate recruitment of basal factors and RNA polymerase II to promoters

2. stimulate activity of already assembled promoters

3. faciliate changes in chromatin structure

How do activator proteins work broadly

By interacting directly or indirectly with basal factors at the promoter or regulatory proteins (at 3 dimensional complex of proteins)

What must typically be present to increase transcription? (protein)

DNA binding protein

What are the two domains in an activator protein? Describe them

Activator domain and sequence-specific DNA binding domain

The activator domain interacts with other transcriptional regulatory proteins, and the DNA binding protein binds to the enhancer sequence

What are the three mechanisms in how transcriptional repressors reduce transcription?

1. repressor protein competes with activator protein for an enhancer

2. repressor protein either blocks the DNA binding domain or the activation domain of an activator protein (both are needed for the assembly of an activator protein)

3. repressor protein interferes with the basal transcription apparatus

Overall: think of a repressor as something that tries to get in the way of everything

What is the effect of transcriptional repressors on transcription?

They reduce transcription

True or false: each GAL gene has its own promoter in eukaryotes

True

What are GAL genes responsible for? In what conditions are they expressed?

Encoding enzymes needed to metabolize galactose.Therefore, they are only expressed if galactose is present

what does galactose induce (genetics-wise/structural GAL genes)

the 100-fold expression of four structural genes: GAL1, GAL2, GAL17, and GAL10

What are the three regulatory GAL genes involved in GAL gene transcription

GAL4, GAL80, and GAL3

What controls the transcription of each structural gene in GAL system

By an enhancer/upstream activator sequence (UASg)

What is the purpose of GAL4, GAL80, and GAL3?

GAL4: transcriptional regulator that binds to UASg (upstream activator sequence); it has an activation domain and a DNA binding domain (activator protein); when it is bound to a UASg, maximal transcription occurs of GAL genes

GAL80: a repressor that binds to the activation domain of GAL4

GAL3: binds to GAL80 to inhibits its interaction with GAL4

Describe how GAl4, GAL80, and GAL3 are connected

• GAL4 on its own when bound to a UASg yields maximal transcription howeverrr

• GAL80 binds to GAL4 to inhibit its ability to activate transcription

• GAL3: binds to GAL80 to prevent it from interacting with GAL4

What regulates GAL3 and GAL80 binding?

Galactose (serves as effector)

Describe GAL3 and GAL80 activity in the presence and absence of galactose?

Presence: GAL3 will bind to GAL80 and prevent it from inhibiting GAL4, so transcription of GAL genes is allowed by GAL4 stimulating the basal apparatus

Absence: GAL3 will not bind to GAL80, so GAL80 will bind to GAL4 and inhibit transcription

Overall: allows for the transcription of GAL genes only when needed/when galactose is present

Describe/predict whether or not transcription would be allowed or inhibited (assuming galactose is present) for the following mutations

• mutation in GAL4 that affect activation domain of GAL4 protein

• mutation in GAL4 that effect DNA binding domain of GAL4

• mutation is UASg that affects binding of GAL4

• mutation in GAL80 that blocks binding of GAL80 and GAL4 protein (galactose is not present)

• mutaiton in GAL80 that affects binding of GAL3 to GAL80

• mutation in GAL3 that affects binding of it to GAL80

1. inhibits

2. inhibits

3. inhibits

4. allows

5. inhibits

6. inhibits

What is the main difference in transcription and translation among prokaryotes and eukaryotes?

Transcription and translation occur simultaneously in prokaryotes while they occur separately in eukaryotes (hence more opportunity to stop something from occurring

What is alternative splicing? What does it result in?

Alternative splicing is when different sections of a gene are considered exons and introns; different sections aka different genes will then be transcribed, resulting in different proteins. commonly, alternative splicing results in isoforms, which are different forms of the same protein but will have different molecules functions (ex: different DNA binding domains)

How can alternative splicing be regulated?

By trans-acting proteins that interact with the exonic and interonic splicing enhancers, which then influences expression

review: how is sex determined in drosophila? but what is the genotype of females and males?

X:A ratio so 1:1 is female and 1:0.5 is male (between 1 and 0.5 is intersex)

females will be xx and males will be xy

What are the three genes required for different steps in the sex determining pathway? What does each one do/how they are connected

The three genes are the sex-lethal (Sxl), transformer (tra), and the double-sex(Dsx)

sxl: this is a gene transcribed very early in the embryonic stage and is determined by the X to A ratio

the sxl will then control the alternative splicing of the tra pre-mRNA

the tra protein (results from the tra pre-mRNA) will control the alternative splicing of the Dsx pre-mRNA; the Dsx gene→Dsx proteins will then lead to male and female differentiation

For tra pre-mRNA and Dsx pre-mRNA, what is being changed? then what is being influenced?

the pre-mRNA will undergo alternative splicing, and this will ultimately affect what is being transcribed and translated. therefore, this will affect the resulting protein

overall: RNA is being processed differently and this impacts the activity of the protein (and ultimately phenotype with the Dsx proteins)

What is the difference in the expression/functinality of the sxl, tra, and dsx in males versus femlaes

Males: no functional Sxl in embyos→no functional Tra protein→male isoform of Dsx protein

Female: functinoal Sxl in embryo→functional Tra protein→female isoform protein of Dsx

Explain the pathways of gender differentiation with sxl, tra, and dsx with alternative splicing

Females: The XX genotype results in Sxl transcription early in the embryonic stage. This Sxl transcription and ultimately Sxl protein will then influence the alternative splicing of Tra (aka what exon to include/exclude—excludes exon 3 to prevent premature stop codon). The proper tra mRNA (after processing) will then result in the tra protein. This protein is responsible for the alternative splicing of the Dsx pre-mRNA (activates the use of the 3’ splice site). This will result in the female specific mature Dsx mRNA, which ultimately leads to the Dsx-F protein that is active and results in female differentiation (female phenotype)

Males: The XY genotype will result in the delayed activation of the Sxl gene, so the Sxl protein is not active=there will be a premature stop codon in the tra pre-mRNA. Therefore, the tra protein will not be functional, so the 3’ splice site will not be active in the alternative splicing of the Dsx pre-mRNA; this will result in the male-specific mature Dsx mRNA=proper Dsx male protein=male differentiation=male phenotype

Summary of the relationship between Sxl, Tra, and Dsx

the genotype dictates whether the Sxl gene will be active for the Sxl protein. If the Sxl protein is present, this will influence how the pre-mRNA of Tra will be spliced (what is considered an exon). A nonfunctional Sxl results in a nonfunctional Tra and vice versa. A nonfunctional Tra will result in a male Dsx isoform portein while a functional Tra will result in a female Dsx isoform protein. Overall, a change in whether or not a gene is transcribed (Sxl) can influence how others genes are transcribed and translated (Tra and Dsx), which ultimately affects the function of their protein. For Dsx, the proteins will differ on isoform and dictate the phenotype of the Drosophila.

What happens when there are loss of function mutations Sxl and Tra?

The drosophilia will be phenotypically male (because no Sxl=nonfunctional Tra=male isoform of Dsx protein=male phenotype)

What happens when there is a loss of function in Dsx gene

XX and XY flies are intersex

For Tra pre-mRNA, what is present that results in a nonfunctional Tra

an in-frame stop codon

How are the Dsx isoforms (male and female specific) differ/are similar

They differ on activation/dimerization domains at the C-terminus

They have a common DNA binding domain at the N-terminus

Describe cell type, location of function in eukaryotic cells (besides being synthesized in the nucleus), and function: Ribosomal RNA (rRNA)

1. Prokaryotic and eukaryotic

2. Cytoplasm

3. Structural and function components of the ribosome

Describe cell type, location of function in eukaryotic cells (besides being synthesized in the nucleus), and function: Messenger mRNA

1. Prokaryotic and eukaryotic

2. Nucleus and cytoplasm

3. Carries genetic code for proteins (to be translated)

Describe cell type, location of function in eukaryotic cells (besides being synthesized in the nucleus), and function: Transfer RNAs (tRNAs)

1. Prokaryotic and eukaryotic

2. Cytoplasm

3. Processing of pre-mRNA

Where is all eukaryotic RNA synthesized?

Nucleus

What two RNA’s are involved in RNA interference?

MicroRNA (miRNA) and small interfering RNA (siRNA)

Describe cell type, location of function in eukaryotic cells (besides being synthesized in the nucleus), and function: MicroRNA (miRNA)

1. eukaryotic

2. Nucleus and cytoplasm

3. Inhibits translation of mRNA

Describe cell type, location of function in eukaryotic cells (besides being synthesized in the nucleus), and function: Small interfering RNA (siRNA)

1. Eukaryotic

2. Nucleus and Cytoplasm

3. Triggers degradation of other RNA molecules

Describe cell type, location of function in eukaryotic cells (besides being synthesized in the nucleus), and function: Long noncoding RNA (lncRNA)

1. Eukaryotic

2. Nucleus and cytoplasm

3. Variety of functions

What are two RNA-mediated mechanisms that control gene expression? Describe them (not the types of RNA)

1. Co-suppression: discovered with trying to engineer transgenic petunias to increase the intensity of flower color, but they ended up variegated. this shows that the transgene encodes for dsRNA (double-stranded RNA) that triggers RNA interference

2. RNA interference (RNAi): involved double-stranded RNA (dsRNA) that can silence gene expression (seen in C. elegans) (does the interfering rather than doing the triggering seen in co-suppression)

What is RNA interference (RNAi) (definition)

Posttranscriptional regulation that is triggered by double-stranded (dsRNA) but mediated by small noncoding RNAs; it regulates many key developmental processes and plays a protective role in response to viral infection

Where are RNAi’s found?

In all eukaryotes

What are the three major classes of RNAi’s

microRNA’s (miRNA), small interfering RNAs (siRNA), Piwi interacting RNAs (piRNAs)

What is RNAi a useful tool for?

Reverse genetics

How has RNAi evolved?

It has evolved to protect genomes against the mutational effects of transposable elements (DNA sequences that can move and result in mutations).

Many transposons are located in heterochromatin=silent but mutations in RNAi can reactive them by reversing transcriptional silencing

Compare and contrast miRNA and siRNA based on origin, target, size, action, and cleavage of

1. Origin: miRNA is from RNA from a distinct gene while siRNA comes from mRNA, transposon, or a virus

2. miRNA targets genes other than the gene it was transcribed from (so if transcribed from gene B it will affect gene A), but siRNA will only target the genes from which they were transcribed from (transcribed from gene b=affects gene B)

3. they are both small

4. action: miRNA will inhibit translation (remember that miRNA affects other genes that it was not transcribed from) while siRNA will degrade mRNA (makes sense since it can only affect genes from which it was transcribed from)

5. miRNA cleaves single-stranded RNA that forms short hairpins of double-stranded RNA while siRNA cleaves off of RNA duplex or single stranded RNA that forms long hair pins

What are the four ways to regulate gene expression by RNAi?

1. Cleavage of target mRNA by Slicer (known as Arugonate proteins in some species)—siRNA will serve as the recognition site for slicer will slicer does the chopping

2. Inhibition of translation of target mRNA (facilitated by miRNA)

3. Transcriptional silencing of a target gene (siRNA + RITS + methylating enzymes)

4. Slicer-independent degradation of target mRNA (do not worry about this one)

Describe how miRNA and siRNA is processed (aka how it forms with RISC)

miRNA:

1. miRNA gene (DNA) is transcribed, cleavage occurs, and dicer comes in to form a smaller fragment=miRNA

2. double-stranded RNA is recognized by Dicer and produced smaller fragments of siRNA

For both, the smaller fragments will be bound by RNA-induced silencing complex (RISC) where one strand will be retained and the other will be degraded (small fragment of ssRNA is left with RISC)

Explain how siRNA and miRNA function differently

After binding with RISC, they both base pair with their target DNA. However, miRNA imperfectly pairs with the target mRNA to inhibit the tanslation of the mRNA. siRNA perfectly pairs with the target mRNA (especially since it comes from the same gene). Then, cleavage and degradation occur (siRNA recruits sniper though to cleave the mRNA)

How does transcriptional silencing of a target gene work?

siRNA + RITS (different protein complex) bind to the DNA via complementary sequences and attract methylating enzymes. These methylating enzymes can methylate DNA or histones to inhibit transcription (basically turning euchromatin into heterochromatin)

How is RNAi used as a tool in research and therapeutic applicaitons?

-research: RNAi can selectively “knockdown” the expression of selected genes to determine their effect on the phenotype

-therapeutic applications: can use clinically to reduce expression of genes whose overexpression causes certain diseases

What are long noncoding RNAs (lncRNAs) and what are they used for

They are long RNA molecules that do not encode proteins (200 to 100,000 nucleotides long)

While the function of all lncRNAs is unknown, they seem to function in gene expression (ex: Xist lncRNA assists with dosage compensation in mammals)

How do siRNA + miRNA differ from lncRNAs

Mainly on size. There are small details that are unknown since the purpose of lncRNA’s is unknown

What does subcellular localization entail?

Cells can move RNA/push it to a specific location until it is needed there; it does not get translated until it is in the RIGHT location

Why localize mRNA and not protein?

It requires less energy to transport mRNA, it prevents ectopic activity of protein (aka having a protein in the wrong spot), facilitates the formation of macromolecular complexes (high local concentration and simultaneous translation for proteins of multi-protein complex), and refines gene expression specially and temporally