Chapter 15: Gene Regulation in Eukaryotes & Chapter 17: Non-coding RNAs

transcription factor

a category of proteins that influence the ability of RNA polymerase to transcribes DNA into RNA

general transcription factors, regulatory transcription factors

two main types of transcription factors

general transcription factor (GTF)

* one of several proteins that are necessary to initiate basal transcription at the core promoter
* required for the binding of RNA polymerase to the core promoter and for progression to the elongation stage

regulatory transcription factor

a protein or protein complex that binds to a regulatory element and influences the rate of transcription via RNA polymerase

regulatory sequence

a segment of DNA that is recognized by a regulatory transcription factor. The binding of the transcription factor affects the rate of transcription.

activator

a regulatory protein that binds to DNA and increases the rate of transcription

enhancer

a DNA sequence that functions as a regulatory element in eukaryotes. The binding of a regulatory transcription factor to an enhancer increases the rate of transcription.

activator, enhancer

_____ binds to an _____ and increases the rate of transcription

repressor

a regulatory protein that binds to DNA and inhibits transcription

silencer

a DNA sequence that functions as a regulatory element in eukaryotes. The binding of a regulatory transcription factor to a silencer decreases the rate of transcription.

repressor, silencer

_____ binds to a _____ and decreases the rate of transcription

combinatorial control

the phenomenon widely observed in eukaryotes in which the combination of many factors determines the expression of any given gene

domain

a segment of a protein that has a specific function

binding, small effector

one domain of a transcription factor may have a DNA-_____ function, and another may provide a binding site for _____ _____ molecule

motif

in proteins, the name given to an amino acid sequence that has a very similar structure and function in many proteins

helix-turn-helix motif, helix-loop-helix motif, zinc finger motif, leucine zipper motif

structural motifs found in transcription factor proteins

helix-turn-helix motif

* two alpha helices are connected by a turn
* the alpha helices bind to the DNA within the major groove

helix-loop-helix motif

* a short alpha helix is connected to a longer alpha helix by a loop
* a dimer is formed from the interactions of two helix-loop-helix motifs, and the longer helices are binding to the DNA

zinc finger motif

* each since finger is composed of one alpha helix and two antiparallel beta sheets
* a zinc ion (Zn 2+) holds the zinc finer together

leucine zipper motif

* the leucine zipper promotes the dimerization of two transcription factor proteins
* two alpha helices (coiled coil) are intertwined due to interactions between their leucines

alpha helix, major groove, recognition helix, hydrogen, positively, negatively

helix-turn-helix and helix-loop-helix motif

* the protein secondary structure known as a _____ _____ occurs frequently in transcription factors
* alpha helix is the proper width to bind into the _____ _____ of the DNA double helix
* the _____ _____ makes contact with and recognizes a base sequence along the major groove of the DNA
* _____ bonding between the amino acid side chains in an alpha helix and the nucleotide bases in the DNA
* recognition helix often contains many _____ charges amino acids (ex: arginine and lysine) that favorably interact with the DNA backbone, which is _____ charged

alpha, beta, zinc, major

zinc finger motif:

* composed of one _____ helix and two _____ sheets that are held together by a _____ metal ion
* can also recognize DNA sequences within the _____ groove

leucine

leucine zipper motif:

* dimerization and DNA binding of two proteins that have several _____ amino acids (a zipper)
* alternating leucines in both proteins interact (“zip up”), resulting in protein dimerization

homodimer

a combined structure (a dimer) formed when two polypeptides encoded by the same gene bind to each other

heterodimer

a combined structure (a dimer) formed when two polypeptides encoded by different genes bind to each other

up regulation

genetic regulation that leads to an increase in gene expression

down regulation

genetic regulation that leads to a decrease in gene expression

enhancer, up

_____ increases the rate of transcription: _____ regulation can be 10-to-1000 fold

silencer, down

_____ decreases the rate of transcription: _____ regulation

orientation-independent, bidirectional

many regulatory elements are _____ or _____

orientation-independent

refers to certain types of genetic regulatory elements that can function in the forward or reverse direction

TFIID

a type of general transcription factor in eukaryotes that is needed for RNA polymerase II function. It binds to the TATA box and recruits RNA polymerase II to the core promoter.

activator, help, enhance, coactivators

_____ proteins can enhance the ability of TFIID to initiate transcription

* _____ TFIID bind to the TATA box
* _____ the function of TFIID in a way that facilitates its ability to recruit RNA polymerase II
* exert their effects by interacting with _____

coactivator

an activator protein that increases the rate of transcription but does not directly bind to the DNA itself

repressor, prevent

_____ proteins inhibit the function of TFIID

* _____ the binding of TFIIF to the TATA box
* _____ the ability of TFIID to recruit RNA polymerase II to the core promoter

small effector, protein-protein, covalent

the functions of regulatory transcription factor proteins are controlled in three common ways:


1. the binding of a _____ _____ molecule
2. _____-_____ interactions
3. _____ modifications

* glucocorticoid, diffusing, glucocorticoid, HSP90, NLS (nuclear localization signal), glucocorticoid, homodimer, homodimer, GRE (glucocorticoid response element)

HSP 90 regulation:

* _____ hormone enters the cytosol of a cell by _____ through the plasma membrane
* hormone binds to _____ receptor
* _____ is released from the receptor, thereby exposing a _____
* two _____ receptors form a _____ and then travel through a nuclear pore into the nucleus
* the _____ binds to a _____, which activates the transcription of the adjacent gene, eventually leading to the synthesis of the encoded protein

CREB protein

a regulatory transcription factor that becomes activated in response to specific cell-signaling molecules that cause the synthesis of cAMP

cAMP response element-binding protein

CREB is an acronym for

cAMP response element (CRE)

a short DNA sequence found near certain eukaryotic genes that is recognized by the cAMP response element-binding (CREB) protein

5’-TGACGTCA-3’

consensus sequence of CRE

G, adenylyl cyclase, adenylyl cyclase, cAMP, cAMP, kinase A, kinase A, CREB, CREB, CBP (CREB-binding protein)

CREB regulation:

* extracellular signaling molecule binds to plasma membrane receptors
* when the signaling molecule binds to the receptor, it activates a _____ protein that subsequently activates the enzyme _____ _____
* the activated _____ _____ catalyzes the synthesis of _____
* the _____ molecule then binds to a second enzyme, protein _____ _____, and activates it
* protein _____ _____ travels into the nucleus and phosphorylates several different cellular proteins, including _____ protein
* this phosphorylation activates _____ protein by allowing it to bind to _____, which functions as a coactivator
* this binding leads to the transcription activation of RNA polymerase

chromatin remodeling

changes in chromatin structure that are due to the action of chromatin-remodeling complexes and affect the positions and/or compositions of nucleosomes

closed conformation

a conformation of chromatin that cannot be transcribed and may be tightly packed

open conformation

a loosely packed structure of chromatin that can be transcribed

histone methylation, histone deacetylation, corepressor complexes

euchromatin → heterochromatin

coactivator complexes, loss of H1, histone modifications

heterochromatin → euchromatin

positions, few, spacing, long

change in nucleosome position:

* change in the relative _____ of a _____ nucleosomes
* change in the _____ of nucleosomes over a _____ distance

gaps

eviction of histone octamers:

* remodelers may evict histone octamers from the DNA, thereby creating _____ where nucleosomes are not found

standard, variants

replacement with histone variants:

* remodelers may change the composition of nucleosomes by removing _____ histones and replacing them with histone _____

amino

over 50 different enzymes modify the _____-terminal tails of histones

histone acetyl transferase (HAT)

adds acetyl groups to lysine residues

reduces, decondenses

adding acetyl groups to lysine residues → _____ the positive charge on the histones → _____ the chromatin

histone deacetylases (HDAC)

removes the acetyl groups from histone

increases, condenses

removing the acetyl groups from histone → _____ the positive charge on the histones → _____ the chromatin

chromatin immunoprecipitation sequencing

ChIP-Seq is an acronym for

nucleosomes, variants, covalent

ChIP-Seq allows determination of:

* where _____ are located
* where histone _____ are found
* where _____ modifications of histones occur

formaldehyde, MNase, linker, antibodies, beads, supernatant, pellet, broken, gel electrophoresis, linkers, PCR, DNA sequencing

steps of ChIP-Seq experiment:

* cells are treated with _____, which covalently link proteins to the DNA (crosslink)
* cells are broken apart, and exposed to _____, which cuts the _____ regions between nucleosomes
* DNA that is cross linked to proteins generally remain intact
* add _____ that recognize the core histone proteins
* the antibodies are attached to large _____
* centrifuge that causes formation of _____ (DNA and proteins not attached to the beads) and _____ (DNA and proteins attached to the beads)
* crosslinks between the histones and DNA are _____
* purify fragments of DNA that are \~150 bp in length by _____ _____
* add _____ to the ends of the DNA
* amplify by _____, using primers that are complementary to the linkers, and subject to _____ _____

what ChIP-Seq measures?

what ChIP-Seq measures?

* The end result of ChIP-Seq is a large collection of DNA sequences. These sequences can be matched up with the sequences in the genome to determine which segments in a genome are found within nucleosomes.
* Can determine the relative positions of nucleosomes from the beginning of a gene to the end

DNA methylation

a regulatory mechanism in which an enzyme covalently attaches a methyl group (-CH3) to a base in DNA

cytosine

DNA methylation happens on _____

little

yeast and Drosophila have _____ detectable methylation of their DNA

abundant

DNA methylation in vertebrates and plants is relatively _____

2-7%

in mammals, approximately _____ of the DNA is methylated

DNA methyltransferase

the enzyme that attaches a methyl group to cytosine in eukaryotes

full methylation

methylation of the cytosine in both strands

hemimethylation

methylation of the cytosine in only one strand

CpG islands

in vertebrates and plants, many genes contain _____ _____ near their promoters

CpG islands

a group of CG base sequences that may be clustered near a promoter region of a gene

1000-2000

CpG islands are commonly _____ bp in length

housekeeping gene

a gene that encodes a protein required in most cells of a multicellular organism

unmethylated

in housekeeping genes, the cytosine bases in the CpG islands are _____

tissue-specific gene

a gene that is highly regulated and is expressed only in a particular cell type

silenced, methylation

in tissue-specific genes, the expression of such genes may be _____ by the _____ of CpG islands

active

unmethylated CpG islands are correlated with _____ genes

suppressed

methylated CpG islands are correlated with _____ genes

prevent

methylated CpG islands _____ the binding of an activator protein to an enhancer element

methyl-CpG-binding proteins

a protein that binds to a CpG island when it is methylated

closed, inhibit

methyl-CpG-binding proteins recruit other proteins that change the chromatin to a _____ conformation and thus _____ transcription

Encyclopedia of DNA Elements

ENCODE is an acronym for

iron regulatory protein (IRP)

a translational regulatory protein that recognizes iron response elements that are found in specific mRNAs. It may inhibit translation or stabilizes the mRNA.

iron response element (IRE)

a sequence in mRNAs that is recognized by the iron regulatory protein (IRP)

5

the ferritin-encoding mRNA has an IRE in its _____’-UTR (beginning)

IRP, IRE, prevents

ferritin:

low iron levels → _____ binds to _____ → IRE/IRP complex _____ the translation of the ferritin mRNA

IRP, prevents

ferritin:

high iron levels → iron binds directly to the _____ and causes a conformational change that _____ IRP from binding to IRE → ferritin mRNA is translated to make more ferritin proteins

3

the transferrin receptor mRNA has an IRE in its _____’-UTR (end)

IRP, IRE, stability, promotes

transferrin receptor:

low iron levels → _____ binds to _____ → enhances the _____ of the mRNA → mRNA can be translated to make more transferrin receptor proteins → _____ the uptakes of more iron into the cell

IRP, prevents, degraded, decrease, prevent

transferrin receptor:

high iron levels → iron binds to _____ → _____ IRP from binding to IRE → mRNA is _____ → _____ in the amount of transferrin receptor → _____ the uptakes of too much iron into the cell

translated, not translated

iron is high → ferritin mRNA is _____ & transferrin receptor mRNA is _____

not translated, translated

iron is low → ferritin mRNA is _____ & transferrin receptor mRNA is _____

DNA, mRNA, proteins, small molecules, multiple

ncRNA binding:

* ncRNAs can bind to different types of molecules: (four common types of molecules that are recognized by ncRNAs)
* _____ binding sites

length

ncRNAs are broadly categorized according to their _____

long non-coding RNA (lncRNA)

an ncRNA that is longer than 200 nucleotides

small regulatory RNA (short ncRNAs)

a non-coding RNA that is shorter than 200 nucleotides

microRNA

a non-coding RNA that is usually 20-25 nucleotides in length

microRNA (miRNA), short interfering RNA (siRNA)

the two types of RNAs that promote interference

microRNA (miRNA)

* ncRNAs that are transcribed from eukaryotic genes that are endogenous (normally found in the genome)


* silence the expression of mRNAs via RNA interference

small interfering RNA (siRNA)

* ncRNAs that originate from sources that are exogenous (not normally made by cells)
* silence mRNAs via RNA interference

2000

in humans, nearly _____ genes encode miRNAs

60%

_____ of human protein-encoding genes are regulated by miRNAs