GENETICS 3

What is the difference betwen constitutive and regulated transcription?

What is one advantage and one disadvantage of regulating gene expression?

regulating gene expression is less metabolically costly, and only express genes when you need them, however necessitates accurate sensing of the enviornmental conditions

What is a repressor protein? What is its allosteric site? 

What is an inducer? What is a corepressor? 

What is an activator protein? 

proteins that have a DNA binding proteins. They block transcription initiation and occupy the same space where RNA pol. would normally bind or prevent formation of the open promoter complex.

 What is an effector? What is an allosteric inhibitor? 

allosteric inhibitors prevent binding

What are operons? What are two advantages of operons?

operons are groups of genes and their shared regulatory region. These groups of genes are almost always involved in the same metabolic pathways.

What conditions trigger expression of the lac operon? What conditions prevent expression in the lac operon?

permease must be present to import lactose into the cell. Beta-galactosidase breaks down lactose into monosaccharides. Transacetylase is not required for lactose utilization, but it protects the cell from potentially damaging byproducts of lactose metabolism.

if glucose is present, lactose will not be expressed (turned off) because the cell does not want to waste energy when glucose is the preferred source for bacteria

What is a polycistronic mRNA?

three genes transcribed together: Permease (lacY), Beta-galactosidase (b-gal) and transacetylase (lacA)

 In the lac operon: What is the operator? What binds it? What is the consequence of this? When is it bound? 

the lac operon contains an operator region where the repressor protein binds. This binding prevents RNA polymerase from transcribing the downstream genes. It is bound when lactose is absent, inhibiting gene expression.

In the lac operon, what binds the repressor protein and what is the consequence of this? 

The repressor protein binds to the operator, preventing RNA polymerase from transcribing the downstream genes. This occurs when lactose is absent, keeping the operon off.

 How is allolactose formed? How does lactose enter the cell if the operon is off? 

allosteric domain binds allolactose. if this is bound, the repressor releases DNA. It can be produced from B-gal acting on lactose and can also be broken down into glucose and galactose by B-gal. If alloctose is not present, the repressor will bind the operator and block transcription inititation.

gene expression is never completely zero. there is always some low level of permease and B-gal present. without this, the system would not work

When does cAMP bind CAP? What is the consequence of this? 

in the absence of glucose, the concentration in the cell of cyclic AMP (cAMP) increases. This is caused by activaton of adenylate cyclase, which converts ATP to cAMP. When glucose is present, this enzyme does not catalyze the reaction. 

cAMP binds to CAP causing it to bind to DNA at the CAP binding site. this facilitates RNA polymerase binding and activates transcription.

What is the difference between basal and leaky expression? 

leaxy expression arises as a consequence of repressor binding being reversible and not 100% efficient → low amount of transcription

Basal expression arises as a consequence of a lack of expression and activation.

What types of mutations lead to constitutive mutants? Which act in cis? Which act in trans?

Constitutive mutants express the operon inrrespective of the presence or absence of lactose and glucose. This can happen if there is a mutation in operator that makes it such that repressor can no longer recognize it.

Operator mutations are cis-acting, meaning they only influence transcription of genes on the same chromosome. Coding changes in DNA binding proteins act in-trans. The I+ allele is trans-acting because it is capable of altering expression of genes on other chromosomes.

What types of mutations lead to non-inducible mutants? Do they act in cis or in trans? 

super repressor mutations make the operon unresponsive to induction by allolactose. Mutations in I that alter its allosteric domain without altering its DNA binding properties. Even when allolactse is present, the protein cannot release the DNA. Is mustations act in trans

Define negative feedback

anabolic operons are typically repressible by the end product

 Contrast typical characteristics of catabolic and anabolic operons. 

catabolic operons like the lac operon, are often inducible by the presence of a particular nutrient. Anabolic operons are typically repressible by the end product (negative feedback)

What is attenuation? What is an advantage of attenuation compared to a simple on/off mechanism? 

attenuation is a property of some repressible operons allowing the magnitude of expression to be tightly controlled in response to various concentrations of the endproduct. Cells have evoled to maintain near constant concentrations of amino acids

What is the function of the 1-2 stem loop? 

What determines whether a 2-3 or a 3-4 stem loop will occur? 

2-3 stem loop (region 2 and 3 forming a stem loop) and 3-4 step loop are the most important for attenuation. The 2-3 is mutally exclusive and the 3-4 stem loop signals transcription termination

The formation of the 2-3 or 3-4 stem loop is determined by the concentrations of the amino acids, influencing whether the ribosome pauses at the attenuator region during transcription.

Suppose the trp operon had its attenuator codons mutated to stop codons, what would be the expected result? 

Explain how alternative sigma factors can give rise to genome wide alterations in gene expression.

Explain how translational regulation for ribosomal proteins functions. Is this negative feedback? Why or why not? 

one of the products of each operon can bind their own mRNA at the shine-salgarno seq. preventing their translation. This provides negative feedback and is the main way production of these proteins is regulated. If we have a lot of proteins, we stop making more of them.

Explain how antisense RNAs can block translation. 

if antisense RNA binds to an mRNA it can prevent its translation. Bacteria can tolerate low expression, but high expression can cause problems if genes are getting disrupted by the transposon. The gene has two promoters oriented in opposite directions. P-in is a weak promoter, this drives expression of the transposase. P-out promter is a strong promter and drives expression of an overlapping antisense RNA. When these RNAs base pair the shine-salgarno seq. and start codon are sequestered and cannot bind the ribosome

 Compare and contrast tryptophan’s role as a co-repressor in the trp operon and GlcN6P’s role in regulating glmS. How are these mechanisms similar? How are they different? 

Tryptophan acts as a co-repressor by binding to the trp repressor, enabling it to attach to the operator and inhibit transcription of the trp operon. In contrast, GlcN6P binds to the glmS mRNA and promotes its self-cleavage, thereby regulating its own synthesis. Both mechanisms provide feedback to regulate gene expression in response to metabolite levels, but they do so through different molecular interactions.

Give two reasons why gene regulation is more complicated in eukaryotes as compared to bacteria.

• chromatin states can alter gene expression

• regulatory sequences can be “long range” rather than just in the promoter

What two types of regulation are present within multicellular eukaryotes that are not present in bacteria? Explain what is meant by each

• temporal (during development)

• spatial (tissue specific)

Compare and contrast the characteristics of the core promoter with that of an enhancer sequence.
How are they similar and how are they different?

core promoter

• region that is upstream of the transcription start site

• binds general transcription factors and RNA pol II

• often contains a TATA box

enhancers and silencers

• enhancers increase the expression of target genes

• Silencers down-regulate target genes

• not constrained to be upstream of genes

• can be intronic or exonic, close or far

• enhancer/silencer mosules have many binding sites for different TFs

• this allows them to integrate the activities of different transcription factors to produce different outputs

• can turn genes on or off and can turn gene expression up or down

• cis acting sequences bind trans-acting proteins

Explain how different patterns of SHH expression are accomplished in the brain and in the limbs

Different expression patterns are needed because they have different functions. There is a long-range enhancer that is limb-specific and there is a short range enhancer for the brain

• different transcription factors present = different patterns of expression in different tissues

Propose a plausible outcome for using CRISPR to replace the cobra SHH enhancer with the mouse SHH enhancer within a cobra

took normal mouse limb specific enhancer and used crispr to replace it with the cobra genome. This caused a mouse to develop without limbs. This suggests that morphological evolution is largely driven by differential regulation of genes

Summarize the general argument for why morphological evolution is presumed to more often be driven by regulatory rather than coding changes. Illustrate this argument using the example of humans and chimps

regulatory mutations that do not alter coding sequences can cause large morphological changes .oolkit genes direct development by changing the timing and amount of these genes and drastic differences can arise. humans and chimps share ~99% of amino acid identitiy in protein sequence. genes are regulated in different amounts at different times during devleopment.

A mutation in the beta-globin LCR causes Thalassemia. Propose a plausible mechanism of action of this mutation.

The mutation in the beta-globin Locus Control Region (LCR) leads to decreased transcriptional activation of the beta-globin gene, resulting in reduced production of hemoglobin and the development of Thalassemia. This occurs because the LCR is crucial for enhancing the expression of the beta-globin gene.

Define modularity in the context of enhancers and silencers. Suppose that enhancers were not modular, how would this change the consequences of mutations in these regions?

modularity means that components of an enhancer module can be altered without altering the effects of other componenets.

Why are some non-coding enhancer elements so highly conserved?

Some non-coding enhancer elements are highly conserved because they play critical roles in regulating gene expression across different species, ensuring that essential developmental and physiological processes are maintained.

How can the property of modularity give rise to different species using different binding sites and different trans acting proteins to regulate a gene, but the pattern of gene expression remains similar between these species?

Modularity allows different enhancers to interact with various transcription factors, enabling diverse regulatory combinations while maintaining conserved gene expression patterns across species.

Explain the mechanism of activation of the Gal genes in yeast. Your answer should explain the roles of UASG Gal80, Gal4, and Gal3 both in the presence and in the absence of galactose

Gal1, Gal2, Gal7, and Gal10 genes are needed for galactose import and utilization as a carbon source. Each of these genes has its own promoter, but is regulated by the transcription factor, Gal4. Gal4 binds an enhancer element called an upstream activator sequence (UAS). Gal4 is constitutively expressed, but is bound to Gal80 (also constitutive) and cannot activate transcription in the absence of galactose.

in the absence of galactose, gal 3 is present in the cytoplasm. in the presence of galactose, gal3 moves to the nucleus, where it binds gal8- and causes the release of gal80 from gal4. This allows for activation of gal genes because the activation domain is no longer obscured

Explain the mechanism of repression of the Gal pathway in yeast. Your answer should explain the roles/location of Mig1 both in the presence and absence of glucose

Mig1 is made in the presence of glucose. It binds a silencer between UAS and the gal genes. This recruits another protein called Tup1, and this complex interferes with the Gal4 enhancer element.

Upon glucose depletion, Mig1 is phosphorylated and exported from the nucleus.

Compare and contrast the gal pathway with the lac operon. How are they similar and how are they different?

Both the gal pathway and the lac operon are regulatory systems controlling gene expression in response to sugar availability. They share mechanisms like the use of specific transcription factors (Gal4 for gal genes and LacI for lac operon) and the presence of enhancer or silencer elements. However, they differ in their sugars (galactose vs lactose), regulatory proteins (Gal80/Gal3 in the gal pathway vs LacI in the lac operon), and the differing responses to glucose availability.

How are enhancers directed to only act to increase the transcription of specific target genes?

Insulator sequences bind proteins and direct enhancers to interact with the intended promoter. This blocks communication between enhancers and other promoters. There can be a preference for one enhancer over another. Mutations in insulator sequences can yield inappropriate enhancer activation.

Identify each of the following histone marks as either generally heterochromatic or euchromatic: H3K27me3, H3K9ac, H3K9me3, H3K4me

H3K27me is a repressive mark in facultative heterochromatin.

H3K9me3 is a repressive mark in constitutive heterochromatin

K3K9ac and H3K4me are euchromatic marks

• they alter chromatin structure

• they are transmissible during cell division (mitotically stable)

• they are reversible

• they do not alter DNA sequence

Histone deacetylases remove acetyl groups from histones. Would we expect greater activity of these enzymes in a particular genomic region to increase or decrease transcriptional activity of that region and why?

Greater activity would decrease transcriptional activity because histone deacetylases promote tighter chromatin structure, leading to reduced gene expression.

Contrast nucleosome sliding with nucleosome ejection

nucleosome sliding: nucleosome remains bound but is displaced such that the regulatory sequences are exposed

nucleosome rejction: removal of the nucleosome and moving it to another sequence, exposing the regulatory sequence

Explain the regulation of PHO5 in yeast in as much detail as possible

PHO5 encodes a phosphatase that is repressed in the presence of excess phosphate. It is activated upon phosphate starvation.

PHO4 encodes a protein that facilitates regulation. Under high phosphate conditions, this protein is localized to the cytoplasm. under low phosphate conditions, this protein is localized to the nucleus.

In high phosphate conditions, the TATA box is not exposed and is blocked by the -1 nucleosome. UASp2 is not exposed and is blocked by the -2 nucleosome. UASP1 is bound by Pho2 (transcription activator). Pho2 is bound by NUA4, an acetylase. Promoter histones are not acetylated

In low phosphate conditions, Pho4 is present and binds Pho2. NUA4 acetylates nearby histones, opening the chromatin. Pho2/Pho4 complex displaces the -2 nucleosome, another molecule of Pho4 binds, and the SWI/SNF complex is recruited.

What are lncRNAs and what is their role in X inactivation?

IncRNAs are long noncoding RNAs. They play a key role in gene regulation, X inactivation. Inactivated Xs have only ~12 genes that escape inactivation. One gene is Xist, which is active on the inactivated chromosome and inactive on the activated chromosome. This gene encodes an lncRNA that coats only the X chromosome from which it was derived. This is its only function. This binding facilitates HDACS and methylases to the chromosome, condensing it to a Barr body and rendering it inactive

Define allele specific expression and genomic imprinting.

Most genes do not show allele-specific expression. Both the maternally and paternally derived genes are expressed equally.

Genome imprinting is when either the maternal or paternal alleles are rendered transcriptionally during silent gamete formation. This is usually accomplished by a sex-specific pattern of DNA methylation (an “imprint”), usually a repressive mark.

Explain how IGF2 expression from exclusively the paternal allele is accomplished

On the maternal chromosome, ICR (imprinting control region) is not methylated and can be bound by an insulator protein. A distant enhancer promotes transcription of the maternal H19 allele, not the IGF2 allele.

On the paternal chromosome, ICR and H19 are methylated, and no insulator binds. Long-range enhancer acts on the IGF2 promoter; paternal IGF2 is expressed, but H19 is not. 

If imprinting is disrupted, improper development occurs! altering appropriate gene dosage!

Explain the argument for sexual conflict in the evolution of maternal IGF2 imprinting in species with litter sizes greater than one and common multiple paternity

The system is likely a remnant of sexual conflict that arose due to male and female reproductive strategies being different.

IGF2 promotes the growth of the fetus - it promotes higher provisioning of maternal resources.

Males benefit most from having each of their offspring use as many maternal resources as possible.

Females benefit most from equal partitioning of resources to all of her partitioning of resources to all of their offspring (and not provisioning offspring with too many resources.

Contrast the production of siRNAs and miRNAs

RNA interference is gene silencing mediated by dsRNA molecules, blocking expression post-transcriptionally and blocking transcription itself.

dsRNA has 3 sources:

• bidirectional promoters (produce siRNA, small interfering RNA)

• miRNA (micro RNA) genes fold back on themselves

• dsRNA from a virus

What is the role of dicer in RNAi?

cuts dsRNA is cut into 21-25 bp fragments, acting as a molecular rule

What are two possible outcomes of siRNA or miRNA binding to RISC and then binding a complementary mRNA that would lower expression post transcriptionally?

Processed siRNA or miRNA, then associates with the RISC complex. One strand is degraded, and complementary mRNAs are destroyed, or complementary mRNAs are bound and prevented from being translated. This outcome is determined by how much sequence identity there is between the bound RNA and the mRNA.

Explain the RITS mechanism for how RNAi can alter transcription.

RITS complex carries the siRNA to the nucleus, where is can direct the complex to complementary nascent RNAs. When base pairing occurs, the complex recruits histone-modifying enzymes that close chromatin and spread heterochromatin. This is a mechanism that is extremely important for silencing centromeric repeats in S. pombe

What is the likely reason that RNAi evolved? What is one piece of evidence that supports this?

Current thinking is that RNAi evolved as a defense mechanism against the mutational effects of transposable elements. Mutations in the RNAi machinery can cause activation of normally silent transposable elements.