Molecular Genetics Chapter 15

DNA methylation process

(1) Forms 5'-methylcytosine (2) Hemimethylated or fully methylated

CpG islands are associated with which of the following? A)Nucleosome location B)DNA methylation C)Steroid hormone activity D)cAMP pathway

B)DNA methylation

What activates CREB? A)Binding of cAMP B)Phosphorylation C)Dimerization D)Lowering of cAMP concentration E)Binding to GRE

B)Phosphorylation

Which of the following recognizes response elements, control elements, and regulatory elements? A)General transcription factors B)Regulatory transcription factors C)Mediator D)Transactivation domains

B)Regulatory transcription factors

What might happen if the insulator between two genes is missing? A)Without the insulator, neither gene can be transcribed. B)Transcription factors influencing one gene's expression may abnormally influence a neighboring gene's expression. C)Transcription factors will not be able to influence the expression of either gene. D)Without insulation, both genes will be expressed at maximal levels. E)Transcription factors will not be able to bind to the insulator.

B)Transcription factors influencing one gene's expression may abnormally influence a neighboring gene's expression.

Which one of the following directly interacts with the DNA as a transcriptional regulator? A)cAMP B)G protein C)Protein kinase A D)CREB protein dimer E)Glucocorticoid response element

D)CREB protein dimer

A mutation in which of the following would result in little or no effect on the expression of a gene regulated by a CRE? A)G protein B)Adenylyl cyclase C)Protein kinase A D)CREB E)Glucocorticoid receptor

E)Glucocorticoid receptor

Classes of regulatory elements

Enhancers - Silencers -

DNA methylation usually activates gene expression. ⊚true⊚false

⊚false

Prior to hormone binding, receptors for steroid hormones are usually found in the nucleus of the cell. ⊚true⊚false

⊚false

Housekeeping genes are unmethylated and active in most cells. ⊚true⊚false

⊚true

If a portion of a transcription factor's domain is structurally similar in a variety of organisms, it is called a motif. ⊚true⊚false

⊚true

Nucleosome location may be changed by a process called ATP-dependent chromatin remodeling. ⊚true⊚false

⊚true

Steroid hormones are an example of an effector which regulates regulatory transcription factor activity. ⊚true⊚false

⊚true

The activity of some transcription factors can be regulated by covalent modifications. ⊚true⊚false

⊚true

When a glucocorticoid hormone binds to the glucocorticoid receptor, HSP90 is released. ⊚true⊚false

⊚true

Gene expression regulation in posttranslational modifications:

- feedback inhibition - covalent modification of proteins

What general transcription factor is most often affected by regulatory transcription factors? A)TFIIB B)TFIID C)TFIIE D)TFIIF

B)TFIID

Regulatory transcription factors act as activator or repressor

activator - binds to enhancer repressor - binds to silencer - decrease transcription rate

Regulatory elements characteristics

- up regulated or down regulated by 1,000 to 10,000 folds - orientation-independent/bidirectional - located within the gene or hundreds to several thousand bases from the gene

Iron concentration control through transferrin receptor mRNA

-IRE is located in 3' untranslated region (3-UTR) (a) Low iron (i) IRP binds to IRE of transferrin receptor mRNA (ii) Translation of transferrin receptor protein is synthesized (b) High iron (i) Iron binds to IRP (inactive) (ii) Degradation of transferrin receptor mRNA by endonuclease -No translation

Which of the following is a region in DNA that binds to a steroid receptor? A)GRE B)IRE C)CRE D)CREB

A)GRE

Modulation of regulatory transcription factors

Binding of an effector molecule - Example: Glucocorticoid receptor (a) Hormone-receptor binding releases HSP90 (heat shock protein) (b) Nuclear localization signal (NLS) is exposed (c) Receptor dimerization (d) Hormone-receptor complex binds to glucocorticoid response element (GRE) (e) Gene expression controlled - Response may be glycogenolysis and immune suppression Protein-protein interactions - Example: Glucocorticoid receptor - Formation of dimers -Homodimer or heterodimer

Histone acetyltransferases are directly involved in which of the following? A)Removal of histones from chromatin B)Movement of nucleosomes C)Chemical modification of histones D)Termination of gene expression E)Insertion of variant histone proteins

C)Chemical modification of histones

Genomic imprinting is a result of ___________. A)nucleosome location B)histone activation C)DNA methylation D)serine to leucine changes in the genetic code

C)DNA methylation

A particular gene has a mutation in its NFR that causes it to bind abnormally tightly to histones. What effect would you expect this mutation has on the expression of this gene? A)The expression of the gene would be abnormally high. B)The effect on gene expression is impossible to predict. C)The gene would encode a different protein instead. D)The expression of the gene would be abnormally low or absent. E)The promoter of the gene will be constantly available to transcription factors and RNA polymerase.

D)The expression of the gene would be abnormally low or absent.

Histone code hypothesis

Transcription is in part regulated by chemical modifications of histone proteins (a) Pattern of histone modifications acts like a code or language (b) Covalent modifications are on amino-terminal tails of histone (c) Histone modification directs certain alterations in chromatin structure

Some regulatory transcription factors recruit proteins to the promoter changing _____ _____.

chromatin arrangement

Regulatory transcription factors combinatorial control

gene expression is controlled by multiple regulatory transcription factors - protein-protein-interaction and covalent modification - modification of nucleosomes - DNA methylation

Insulator is a segment of DNA that functions as a boundary between two _____.

genes 1) Insulators as a barrier (a) Barrier to chromatin-remodeling or histone-modifying enzymes (b) Block the effects of enhancers of neighboring genes (c) Loop formation to block the effects of enhancers of neighboring gene

Regulatory transcription factors influence on the ability of RNA polymerase binding to gene and its expression

helps binding of RNA polymerase to the promoter - regulate gene transcription rate

Regulatory transcription factors DNA binding domain

'Motif': group of proteins that has similar DNA binding structures (a) Helical structures are common - recognition helix (b) Bind to major groove of double-stranded DNA (c) Amino acids with positively charged functional groups are abundant (d) Functional modification by dimerization - Leucine zipper motif - Homodimer vs. heterodimer

DNA methylation characteristics

(1) In mammals, 2-7% of the cytosine are methylated (2) DNA methylation usually silences gene expression - Barr body is highly methylated (3) Methyl group in major groove hinders protein binding (4) Enzymatic activity of some restriction enzyme is affected by methylation - HpaII and MspI

CpG islands

(1) Located near promoter region (2) Methylation of CpG islands is closely related with gene expression (a) Housekeeping genes -CpG islands are unmethylated (b) Tissue-specific genes -CpG islands are only methylated when those are not expressed

Chromosome structure and enzyme accessibility

(1) Location of nucleosome and histone variation controls gene expression (2) Closed configuration vs. open configuration -For gene expression, closed configuration need to be change into open configuration

DNA methylation is heritable

(1) Methylated DNA sequences are inherited during cell division (a) de novo methylation or demethylation is observed but rare and highly regulated - Can be one way to control specific gene expression during development or tissue specific gene expression (b) Newly made DNA strand is not methylated - Double stranded DNA is 'hemimethylated' (c) Maintenance methylation by methyltransferase on daughter strand - Double stranded DNA is 'fully methylated

Transcriptional silencing through methylation

(1) Methylation of CpG islands inhibits binding of activator (2) Methylated CpG islands recruit methyl-CpG-binding proteins that compact DNA

Chromatin remodeling complexes

(a) Contains DNA translocases - A catalytic ATPase subunit (b) SWI/SNF-family, ISWI-family, INO80-family

Classes of DNA binding protein motifs

(a) Helix-turn-helix motif (b) Helix-loop- helix motif (c) Zinc finger motif - Made of one α-helix and two β-sheet structure held by Zn+ - Steroid hormone receptors (d) Leucine zipper motif - Leucine-leucine dimer formation

Nucleosome positioning

(a) Typical arrangement pattern is observed (b) Functional significance is not clear

Covalent modification of transcription factor

- Addition of a phosphate group - cAMP response-element binding protein (CREB) (a) Initiated by signal transduction through cell membrane proteins (b) cAMP response element (CRE) is a regulatory DNA sequence where CREB binds (c) cyclic-adenosine monophosphate (cAMP) activates protein kinase A (PKA) (d) Protein kinase A activates CREB by phosphorylation of CREB (e) Phosphorylated CREB binds to CRE and control gene expression

Which DNA sequence important for gene expression is usually contained within the NFR? A)Core promoter B)CREB binding site C)Histone binding site D)Transcription termination sequence E)Hormone response element

A)Core promoter

Which of the following is the most likely location of an insulator sequence? A)At the end of a gene B)In the middle of a gene C)At the beginning of a gene D)Between two genes E)Within an intron

D)Between two genes

TFIID and mediator

Regulatory transcription factors and TFIID (a) Regulatory element is close to the gene (b) Activator on enhancer and coactivator recruit TFIID to the promoter -Coactivator works with activator (not DNA binding protein) (c) Repressor protein on silencer inhibits TFIID from binding to the promoter or recruiting other proteins Regulatory transcription factors and mediator (a) Regulatory element is located at a distance (b) Mediator mediates interaction between RNA polymerase II and regulatory transcription factors (c) Mediator binding controls progression of RNA polymerase II to elongation stages

Nucleosome organization of eukaryotic genes

(1) Nucleosome-free region (NFR) (a) No histone present (b) Promoter and transcription termination site are NFR (c) 150bp in length (d) Not sufficient for own gene activation (2) Histone variant H2A.Z (a) Positioned on -1 and +1 nucleosome (b) Facilitates transcription activation (c) Inversely correlated with methylation

Transcription activation

(1) Through modification of histone protein (nucleosome reposition) (a) Compositional change: Replace histone to histone variants (b) Covalent modification: Methylation, acetylation... (c) Repositioning: Relocate nucleosome (2) Activation process (a) Activator binds to enhancer (b) Chromatin remodeling complex formed (c) Preinitiation complex formed (d) Histone modification as needed

ATP-dependent chromatin remodeling

(a) Change in nucleosome position (b) Histone eviction (c) Replacement with histone variants (i) Histone variants are mutated histone proteins (ii) All histones have variants but histone H4 (iii) Variant may reposition nucleosome

Procedure of ChIP-Seq

(a) Treat the cell with formaldehyde to crosslink between DNA and histone of nucleosomes (b) Rupture the cell and treat with micrococcal nuclease which cut linkers from nucleosomes (c) Add histone antibody treated beads to precipitate nucleosomes (d) Isolate linker DNAs or nucleosome DNAs by centrifugation and electrophoresis (e) Add short and known DNA sequence at both ends of isolated DNA (f) PCR for the sequencing of DNA

ChIP-Seq allows for the determination of:

(a) Where nucleosomes are located (b) Where histone variants are found (c) Where covalent modifications of histones occur

Iron regulatory protein (IRP)

- binds to Iron Response Element (IRE) (1) Iron concentration control through ferritin mRNA -IRE is located in 5' untranslated region (5-UTR)

Regulatory transcription factors recognize cis-regulatory elements of gene

- regulatory transcription factors are called 'response elements', 'control elements', or 'regulatory elements' - cis-regulatory elements are similar to operators in prokaryotes - act as activator

Gene expression regulation in translation:

- small RNAs such as miRNA and siRNA - phosphorylation of translational initiation factors - RNA binding proteins

In the presence of abundant iron in the cell, the iron binds IRP. The complex is then released from IREs leading to __________. A) an increase in ferritin mRNA translation and a decrease in transferrin receptor mRNA stability B) an increase in transferrin receptor mRNA stability and a decrease in ferritin mRNA translation C) an increase in transferrin receptor mRNA stability and ferritin mRNA translation D) an decrease in transferrin receptor mRNA stability and ferritin mRNA translation

A) an increase in ferritin mRNA translation and a decrease in transferrinreceptor mRNA stability

Which DNA sequence would also work in place of the following bi-directional enhancer? 5' GTTC 3'3' CAAG 5' A)5' GAAC 3'3' CTTG 5' B)5' CTTG 3'3' GAAC 5' C)5' CAAG 3'3' GTTC 5' D)5' CTTC 3'3' GAAG 5'

A)5' GAAC 3'3' CTTG 5'

Which of the following might complicate the interpretation of ChIP-Seq data? A)Because histones can be moved around or ejected from the chromatin, DNA that is only sometimes wrapped around a histone might not be sequenced. B)DNA wrapped around nucleosomes can never be sequenced. C)The regions of DNA wrapped around a histone is too long to be sequenced with current technologies. D)The DNA between nucleosomes are too compacted to be sequenced.

A)Because histones can be moved around or ejected from the chromatin, DNA that is only sometimes wrapped around a histone might not be sequenced.

How are eukaryotic transcription factors regulated? (Check all that apply.) A)Covalent modification B)Binding to cytoskeletal elements C)Binding of an effector molecule D)Protein-protein interaction

A)Covalent modification C)Binding of an effector molecule D)Protein-protein interaction

Select all structural motifs that promote protein dimerization. A)Leucine zipper B)Helix-loop-helix C)Helix-turn-helix D)Zinc finger

A)Leucine zipper B)Helix-loop-helix

Which of the following is a major finding from the ENCODE project? A)More than 80% of the human genome sequence is linked to a biological function. B)In humans, one gene encodes exactly one protein. C)Genes were identified for all known diseases. D)There are more than 10 million regulatory sites in human DNA.

A)More than 80% of the human genome sequence is linked to a biological function.

In numerous studies that investigate gene expression, the mRNA levels of proteins are examined. If one is interested in the amount of protein being produced, is this the best approach to use? A)No, directly determining the amount of protein produced would be best because many steps are involved in regulating protein levels. B)Yes, this is the best approach because all mRNA is eventually translated into protein. C)No, examining the transport of mRNA from the nucleus would provide more reliable data since mRNA must be transported to the cytoplasm for translation. D)No, examining mRNA processing would be more reliable since that is the limiting step for protein production.

A)No, directly determining the amount of protein produced would be best because many steps are involved in regulating protein levels.

Covalent modification of amino-terminal tails of histone or histone variants

Acetylation Methylation Phosphorylation

Researchers frequently wish to produce proteins in cells in a regulated fashion so that the effects of the protein can be studied under conditions when and when it is not present. Which of the strategies outlined below would be best suited for these types of studies? A)Creating a piece of DNA where the gene for their protein of interest is under the control of a house keeping gene promoter. B)Creating a piece of DNA where the gene for the protein of interest is under control of a glucocorticoid response element. C)Creating a piece of DNA where the gene for the protein of interest lacks any regulatory regions and only has a core promoter. D)Creating a piece of DNA where the gene for the protein of interest is not changed from how it is in the genome.

B)Creating a piece of DNA where the gene for the protein of interest is under control of a glucocorticoid response element.

In ChIP-Seq, the DNA fragments to be sequenced are around 150 bp in length. What does this size of DNA represent? A)The length of a promoter B)The amount of DNA wrapped around a nucleosome C)The average length of a gene D)The average distance between genes E)The amount of DNA between nucleosomes

B)The amount of DNA wrapped around a nucleosome

If a DNA template is being transcribed in vitro what would be the effect on RNA production if an inhibitor of TFIID was added? A)Transcription would stop immediately. B)Transcripts being produced would continue but there would be no new initiation. C)Transcription would stop immediately but there would be one more round of initiation. D)Transcription rates of templates already being transcribed would increase but there would be no new initiation.

B)Transcripts being produced would continue but there would be no new initiation.

In apoptosis, a process of programmed cell death, the cell undergoes controlled degradation of cellular components including nuclear DNA. Researchers have noted that if the DNA is isolated from an apoptotic cell, it appears as distinct bands of multiples of approximately 145 bases in length. Why does this occur? A)There is a specific DNase that digests DNA every 145 base pairs. B)DNA sequences that appear every 145 base pairs allows for easier DNA breakage. C)DNA that is wrapped around the histones is not being degraded. D)DNA is coated with transcription factors every 145 base pairs.

C)DNA that is wrapped around the histones is not being degraded.

What would be the result of a mutation in Hsp90 that prevented its binding to the glucocorticoid receptor? A)Glucocorticoid receptor could not form a dimer. B)The nuclear localization signal would no longer function. C)Expression of the regulated genes would become constitutive. D)The hormone would not be able to bind to the glucocorticoid receptor.

C)Expression of the regulated genes would become constitutive.

A mutation in the iron response protein (IRP) that made it incapable of binding iron would result in __________. A)no expression of either ferritin and transferrin receptor B)constituitive transferrin receptor and ferritin expression C)consituitve transferrin receptor and no ability to stimulate ferritin expression D)an increase in transferrin receptor and ferritin expression

C)consituitve transferrin receptor and no ability to stimulate ferritin expression

A particular cell contains all of the standard histones but lacks several histone variants. Which of the following MAY be true of this cell? A)The cell will be completely unable to remodel its chromatin. B)The cell will have an enhanced ability to chemically modify its histones. C)The cell will be unable to express any of its genes at all. D)The cell will express different sets of genes than other cells in the same organism. E)The cell will not be able to divide.

D)The cell will express different sets of genes than other cells in the same organism.

For which of the following histones has variant forms not yet been identified? A) H1 B) H2A C) H2B D) H3 E) H4

E) H4

Regulation of translation

iron-response element - Uptake of iron into mammalian cells (1) Iron binds to transferrin (2) Iron-transferrin complex binds to transferrin receptor on the membrane (3) Iron-transferrin-receptor complex is taken into the cell by endocytosis (4) Iron is released to cytoplasm (a) Iron may be used in enzymatic reaction or stored in ferritin

A heterodimer occurs when two identical transcription factors interact on a sequence of DNA. ⊚true⊚false

⊚false

A repressor protein would enhance the ability of TFIID to bind to the TATA box of the promoter. ⊚true⊚false

⊚false

A transactivation domain is an example of a motif found in transcription factors. ⊚true⊚false

⊚false

Activator proteins bind to silencer sequences and repressor proteins bind to enhancer sequences. ⊚true⊚false

⊚false

DNA that contains actively transcribed genes would most likely contain chromatin in the closed configuration. ⊚true⊚false

⊚false

Insulators exert their effects on gene expression through histone acetyltransferase, histone deacetylase, and RNA polymerase. ⊚true⊚false

⊚false

Methylation of DNA bases is one way that regulatory transcription factors may influence gene expression. ⊚true⊚false

⊚false