Mol Gen Module 3

What role does RNA stability play in gene regulation? What controls RNA stability in eukaryotic cells?

-if RNA is unstable, it will be degraded and unable to use for translation --> amt of protein synthesized depends on amt of available mRNA -stability controlled by 5' cap, polyA tail, 5' UTR, 3' UTR, and coding region

Describe 3 pieces of evidence that supports the hypothesis that DNA methylation is important factor in gene regulation.

-inverse relationship b/w amt of methylation and degree of gene expression -methylation patterns are tissue specific and heritable -5-azacytidine causes undermethylation and changes gene expression

Explain why mutations in the lacI gene are trans in their effects, but mutations in the lacO gene are cis in their effects.

-lacI gene encodes the lac repressor protein, which can diffuse within the cell and attach to any operator. -lacO gene encodes the operator. It affects the binding of DNa polymerase to the DNa, and affects only the expression of genes on the same molecule of DNA.

How are miRNAs produced? How do miRNAs function to affect production of proteins? Provide sufficient detail in your answer for full credit.

-miRNAs produced when dsRNA is cleaved into small fragments of ssRNA

How can the expression of a gene be altered with an RNA?

-using RNAi, can knock out func of gene -investigators introduce short synthetic dsRNAi into cells -they trigger RNA degradation pathways, which target mRNA for degradation/inhibition

Fill in the blanks in the "level of transcription" column of this table with: + for high levels of transcription, and - for minimal levels of transcription of the lac operon. Consider regulation by both the lac repressor and CAP (catabolite activator protein). The strain is wild type, with no partial diploidy. Please label your answer with numbers 1-4 based on the chart below. Medium conditions Level of transcription 1 high glucose, no lactose 2 no glucose, high lactose 3 high glucose, high lactose 4 no glucose, no lactose

1 - 2 + 3 - 4 -

The following table shows several bacterial strain lac operon genotypes (some are partial diploids). a) Fill in the blanks in the "lactose absent" and "lactose present" columns in this table. (+) means significant levels of active ß-galactosidase enzyme can be detected. (-) means no significant levels of active ß-galactosidase enzyme. Label your answers: Lactose absent #1-6 and Lactose present #1-6 Strain genotype Lactose absent Lactose present 1. lacI+ lacP+ lacO+ lacZ+ lacY+ - + 2. lacI+ lacP+ lacOc lacZ+ lacY+ + + 3. lacI+ lacP+ lacOc lacZ- lacY+ - - 4. lacIs lacP+ lacOc lacZ+ lacY+ + + 5. lacI- lacP+ lacO+ lacZ+ lacY+ / lacI+ - + 6. lacIs lacP+ lacO+ lacZ+ lacY+/ lac I+ - -

1 - + 2 + + 3 - - 4 + + 5 - + 6 - -

Over the past decade, the most significant finding in biology has been the identification of miRNAs and siRNAs and their role in regulating the development of many multicellular organisms. Briefly describe the four different ways these small RNAs influence gene expression. P. 463

1) degrading mRNA through slicer activity 2) inhibiting transcription due to methylation of histone proteins or DNA sequence 3) inhibit translation: miRNAs bind to complementary regions of mRNA 4) silencing genes: miRNA causes slicer-independent mRNA degradation by binding to complementary 3' UTR region

What are three ways in which gene regulation is accomplished by modifying the structure of chromatin?

1) modify histone proteins 2) chromatin remodeling 3) methylate cytosine in DNA

Name six different levels at which gene expression might be controlled. List five levels at which gene control can take place in eukaryotes.

1. Packaging of DNA into chromatin (chromatin structure) and modifications to DNA 2. Regulation of transcription 3. Processing of RNA transcripts 4. Regulation of RNA stability 5. Regulation of translation 6. Control of the activity of proteins after translation

In what part of the mRNA does degradation generally begin?

5' end w/ removal of methyl cap

What are the differences between neutral mutations and silent mutations?

Neutral - changes a.a sequence but not biological function Silent - don't change a.a sequence because of multiple codons for 1 amino acid

What is the difference between positive and negative control? What is the difference between inducible and repressible operons?

Positive control: the regulatory protein is an activator Negative control: the regulatory protein is a repressor Inducible operon: operon is normally off. Something must happen for transcription to be induced, or turned on. Repressible operon: operon is normally on. Something must take place for transcription to be repressed, or turned off.

List some important differences between bacterial and eukaryotic cells that affect the way in which genes are regulated.

Prokaryotes: genes are clustered in operons and expressed through single mRNA, RNA polymerase is either blocked or stimulated by regulatory proteins, no chromatin structure Eukaryotes: genes are separate with own promoter and transcribed on individual mRNA, initiation requires RNA pol, general transcription factors and activators, chromatin remodeling necessary for transcription

What are the basic regulatory elements in a strand of DNA that allow for genes to be regulated? Are these different in eukaryotes and prokaryotes? If so, why?

Promoters, operators in prokaryotes. Promoters, proximal promoter elements, enhancers, and silencers in eukaryotes. Prokaryotic genes are transcribed as operons, while eukaryotic genes are not and, the role of the nucleosome is different in eukaryotic transcription.

The diagram below shows a chromosomal inversion around a gene. The arrows show how the gene will become physically turned around after mutation. Assuming there is a distinct mutant phenotype, describe in detail the type of mutation, including whether it should be dominant or recessive. Would this phenotype have the same effect in a haploid creature as opposed to a diploid one?

Since the promoter will in next to the silencer and this will silence the expression of the genes which will be a loss of function mutation for the allele. In the diploid, only one of the copies will be mutated so this will be recessive while the haploid, since there is only one, the dominant/recessive is essentially meaningless.

Fill in the blanks in the "strain genotype" column of the following table. Write chromosomal genotypes with no partial diploidy. (+) means transcription of the lac operon. (-) means no transcription of the operon. The first line is filled in for reference. Please label your answers 1-3. Include the strain genotype for lacI, lacP, lacO, lacZ, and lacY

Strain genotype Lactose absent Lactose present 1 lacI+ lacP+ lacO+ lacZ+ lacY+ - + 2 lacI+ lacP+ lacOc lacZ+ lacY+ + + 3 lacIs lacP+ lacO+ lacZ+ lacY+ - -

The following table lists several genotypes associated with the lac operon in E. coli. For each, indicate with a "+" or a "—" whether β-galactosidase would be expected to be produced at induced levels. (Assume that glucose is not present in the medium.) Genotype No lactose With lactose a I + O+ Z+/ F' I + O+ Z+ _________ _________ b I - Oc Z -/ F' I - Oc Z- __________ _________ c I - Oc Z +/ F' I - O+ Z+ __________ _________ d I s Oc Z -/ F' I s O+ Z+ __________ _________

a -,+ b -,+ c +,+ d-,-

Match each number with the closest type of DNA:

a. moderately repetitive DNA - rRNA, tRNA b. highly repetitive DNA - telomeres c. short interspersed elements - ? d. long interspersed elements - ? e. unique-sequence DNA - gene encoding sequences

A leading hypothesis regarding the source of some transposable elements is the integration into the genome of a virus. What evidence supports this hypothesis?

conservation of gene sequences b/w transposable elements and viral genes

What is catabolite repression? How does it allow a bacterial cell to use glucose in preference to other sugars?

defn: presence of glucose inhibits transcription of genes involved in metabolism of other sugars -cAMP regulated so its conc is inversely proportional to amt of glucose -when cAMP levels are high, it binds CAP -CAP-cAMP complex binds DNA, increasing efficiency of RNA binding -high rates of transcription/translation of genes that cause glucose production -when cAMP levels low, RNA pol won't bind

P-element transposons provide a powerful tool for the study of Drosophila genetics. What are P elements, and why are they so useful?

defn: transposon specific to Drosophila -probably arose from horizontal transfer -can be used to manipulate Drosophila genome and create genetically modified flies for research

Define RNA silencing (or interference). Explain how siRNAs arise and how they potentially affect gene expression. How are siRNAs different from the antisense RNA mechanism?

defn: when siRNA binds to mRNA, stimulating mRNA degradation -arise from cleavage of dsRNA to make small fragments of ss siRNA -block gene expression by degrading mRNA before it can be translated -antisense RNA binds to mRNA too, but physically prevents translation

A mutant E. coli strain, grown under conditions that normally induce the lac operon, does not produce ß-galactosidase. What is a possible genotype of the cells?

lacI+ lac P- lacO+ lac Z+ lac Y+

The __________ is a type of _________ protein that binds to a region of DNA in the promoter of a gene called the _________ and prevents ______________ from taking place.

repressor, regulator, operator, transcription

In the previous diagram, if there is a mutation in P and I, which of the following is true?

these are mutations that are, respectively, trans and cis acting on lac operon expression

Insulators can block the effects of enhancers only when

they lie b/w enhancer and promoter

A mutation in the gene for the yeast regulatory protein GAL4 causes yeast to grow poorly on galactose. What is the function of GAL4?

transcription activator for galactose-digesting enzyme gene

____________________act as intermediaries between ______________________________ and specific _____ sequences to modify chromatin structure and activate transcription.

transcriptional activators, chromatin remodeling complexes, DNA

When referring to attenuation in regulation of the tryptophan operon it would be safe to say that when there are high levels of tryptophan available to the organism

transcriptional termination is likely

Under the system of genetic control of the tryptophan operon,

when there's no tryptophan in medium, transcription of trp operon occurs at high levels

The table below lists several genotypes associated with the lac operon in E. coli. For each, indicate with a "+" or a "-" whether β-galactosidase would be expected to be produced. (Note: you are analyzing whether or not you see functional β-galactosidase enzyme or not). Please label your answer No Lactose #1-4 and With Lactose #1-4.

β-galactosidase production No Lactose With Lactose 1. I+O+Z+ /F' I-O+Z+ - + 2. I-O+Z+ /F' I-O+Z+ + + 3. I-OCZ+ /F' I-O+Z- + + 4. ISOCZ+ /F' I+O+Z+ + +