Chapter 8 Flashcards
“prokaryotes”
__________ of gene expression allows cells to adapt to environmental changes and is responsible for the distinct activities of differentiated cell types that make up complex organisms
regulation
“prokaryotes”
______________ is the first step in gene expression, and the initial level at which gene expression is regulated
transcription
“prokaryotes”
studies of _____ have provided the model for subsequent investigations of transcription in eukaryotic cells
E. Coli
“prokaryotes”
____ was discovered first in E. coli and RNA polymerase was purified and studied
mRNA
“prokaryotes”
_____________ catalyzes polymerization of ribonucleoside-5’-triphosphates (NTPs) as directed by a DNA template, always in the 5’ to 3’ direction
RNA polymerase
“prokaryotes”
____ has five types of subunits
bacterial RNA polymerase
“prokaryotes”
the _____ is weakly bound and can be separated from the others. it identifies the _______ sites for transcription intiation
Ơ subunit
the correct sites
“prokaryotes”
____ gene sequence to which RNA POLYMERASE binds to initiate transcription
promoter
“prokaryotes”
promoters are ___________ long and are located 10 and 35 base pairs upstream of the transcription start site
6 nucleotides
“prokaryotes”
consensus sequences are the bases most frequently found in different ________–
promoters
“prokaryotes”
“the functional importance of -10 and -35 regions”
genes with promoters that different from the consensus sequences are transcribed ______ efficiently
less
“prokaryotes”
“the functional importance of -10 and -35 regions”
____ binds to both regions
the Ơ subunit
“prokaryotes”
initially, the DNA is not unwound (_________)
closed-promoter complex
“prokaryotes”
the polymerase then unwinds ____ of DNA to form an open promoter complex, allowing transcription
12-14 bases
“prokaryotes”
after addition of about 10 nucleotides ____ is released from the polymerase
Ơ subunit
“prokaryotes”
during elongation, polymerase maintains an _____ region of ___ base pairs
unwound region
about 15 base pairs
“prokaryotes”
High-resolution structural analysis shows that the B and B’ subunits form a crab-claw-like structure that grips the DNA template.
a channel between these subunits contains the _________
polymerase active site
“prokaryotes”
RNA synthesis continues until the polymerase encounters a _______
stop signal
“prokaryotes”
the most common stop signal is a symmetrical inverted repeat of ____________-
GC-rich sequence followed by seven A residues
“prokaryotes”
transcription of the ______ results in a segment of RNA that can FORM A STABLE STEM-LOOP STRUCTURE
this disrupts its association with the DNA template and terminates its transcription
GC-rich inverted repeat
“prokaryotes”
most transcriptional regulation in bacteria operates at _____________
initiation
“prokaryotes”
studies of gene regulation in the 1950s used enzymes involved in ____________
lactose metabolism
“prokaryotes”
the enzymes are only expressed when _____ is present
lactose
“prokaryotes”
three enzymes are involved?? which ones and describe their activities
B-galactosidase cleaves lactose into glucose and galactose
lactose permease transports lactose into the cell
transacetylase inactivates toxic thiogalactosides that are transported into the cell along with lactose
“prokaryotes”
genes encoding these enzymes are expressed as a single unit, called an __________
operon
“prokaryotes”
two loci control transcription:
- ________, adjacent to transcription initiation site
- i (not in the operon) __________________
o operator
i ,,, encodes a protein that binds to the operator
“prokaryotes”
normal i gene product is a _____, which blocks transcription when bound to o
repressor
“prokaryotes”
when ____is present in normal cells, it binds to the repressor, preventing it from binding to the operator, and the gene are expressed
lactose
“prokaryotes”
the lactose operon illustrates the central principle of gene regulation:
control of transcription is mediated by the interaction of ____ proteins with specific ____________
interaction of regulatory proteins
with specific DNA sequences
“prokaryotes”
____ control elements affect expression of linked genes on the same DNA molecule (e.g., the operator)
cis-acting control elements
“prokaryotes”
other proteins can affect expression of genes _______ (e.g., the repressor)
on other chromosomes
“prokaryotes”
the ____ is an example of negative control- binding of the repressor blocks transcription
lac operon
“prokaryotes”
define negative control:
the regulatory protein ( the represssor) blocks transcription
“prokaryotes”
define positive control
regulatory proteins activate rather than inhibit transcription
example of POSITIVE CONTROL in E. coli
- ________ (preferred energy source) represses expression of the lac operon, even if lactose is also present
- this is mediated by a positive control system. _______________. levels of cAmp increases
presence of glucose
if glucose decreases
cAMP binds to the regulatory protein _________ activator protein (CAP)
catabolite
the stimulates CAP to bind to its target DNA sequence upstream of the ______ operon
lac operon
CAP facilitates binding of RNA polymerase to the ____________
promoter
“eukaryotes”
eukaryotic cells have _____ polyermases that transcribe different classes of genes
three RNA polymerase
“eukaryotes”
transcription takes place on chromatin; __________ is important in regulating gene expression
regulation of chromatin structure
“eukaryotes”
eukaryotic RNA polymerases are complex enzymes, consisting of _____ to _____ different subunits
12 to 17
“eukaryotes”
RNA polymerase II synthesizes mRNA and has been the focus of most ________ studies
transcription studies
“eukaryotes”
unlike prokaryotic RNA polymerase, it require initiation factors that (in contrast to bacteria Ơ factors) are not associated with the ____________
polymerase
“eukaryotes”
_____________ are proteins that involved in transcription from polymerase II promoters
general transcription factors
“eukaryotes”
about ____ of the genes in the human genome encode transcription factors, emphasizing the importance of these protiens
10%
“eukaryotes”
promoters contain several different sequence elements surrounding their transcription sites.
the ______ resembles the -10 SEQUENCE OF BACTERIAL PROMOTERS
other include initiator (lnr) elements, TFIIB recongnition elements (BRE), and downstream elements DCE, MTE, and DPE)
TATA box
“eukaryotes”
_________________ are required for initiation of transcription in vitro
five general transcription factors
“eukaryotes”
transcription factor _______ is composed of multiple subunits, including the ______________ and other subunits (TAFs) that bind to the inr, DCE, MTE, and DPE sequences
TFIID
TATA- binding protein (TBP)
“eukaryotes”
several other transcription factors ___, _____, _____, _____) bind in association with the RNA polymerase II to form the preinitiation complex
TFIIB, TFIIF, TFIIE, and TFIIH
“eukaryotes”
within a cell, additional factors are required to initiate transcription. these include ________, a protein complex of more than 20 subunits; it interacts with both general transcription factors and RNA polymerase
mediator
“eukaryotes”
the mediator complex binds to nonphosphorlated CTD of polymerase II and it ___________________________ (CTD, carboxy terminal domain)
release following phosphorylation of CTD when transcription intiates
“eukaryotes”
the phosphorylated CTD then binds ____________ that facilitate mRNA synthesis and processing
elongation and processing factors
“eukaryotes”
___________ trascribes rRNA genes, which are present in tandem repeats
RNA polyermase I
“eukaryotes”
promoters of r RNA gene are recongnized by two transcription factors which recruit _______ to form and initiation complex.
- USF (___________)
- SL1 (___________)
RNA polyermerase I
- upstream binding factor
- selectivity factor 1
“eukaryotes”
Genes for __________,_____, and some of the small RNAs are transcribed by POLYMERASE III.
they are expressed from three types of promoters
tRNA, 5S rRNA
“eukaryotes”
many _____- acting sequences regulate expression of eukaryotic genes
these regulatory sequences have been identified by gene transfer _______
cis-acting
assays
“eukaryotes”
Gene transfer assays:
regulatory sequences are ligated to a _______ gene that encodes an easily detectable enzyme, such as firefly luciferase
the regulatory sequence directs expression of the reporter gene in cultured cells
reporter gene
“eukaryotes”
__________________: regulatory sequences located farther away from the start site. first identified in studies of the promoter of virus SV40.
Enhancers
“eukaryotes”
activity of enhancers does not depend on their _____ from, or _________ with respect to the transcription initiation site
distance from
or orientation
“eukaryotes”
__________ like promoters, function by binding TRANSCRIPTION FACTORS that then regulate ___________
enhancers
RNA polymerase
“eukaryotes”
_______ looping allows for a transcription factor bound to a distanct enhancer to interact with proteins associated with the RNA polymerase/Mediator complex at the promoter
DNA looping
“eukaryotes”
Enhancers account for _____ or more of human genomic DNA, emphasizing the importance of these elements
10%
“eukaryotes”
many mutations linked to human diseases affect ________ rather than protein coding sequences
enhancers
“eukaryotes”
in any given cell type, multiple enhancers work together to ______ individual genes.
regulate
activity of any given enehancer is specific for the ___________________________.
specificity is maintained partly by ____, which divide chromosomes into independent domains and PREVENT ENHANCERS from acting on promoters located in adjacent domain
promoter of its appropriate target gene.
maintained partly by INSULATORS
“eukaryotes”
genomes are divided into discrete chromosomal domains: _________________
topologically associating domains (TADs)
“eukaryotes”
enhancers and promoters within TAD interact frequently with each other, but only rarely with elements ______________
in other domains
“eukaryotes”
the main protein that binds insulators in vertebrates is __________
CTCF
“eukaryotes”
____________________: like sp1, bind regulatory DNA sequences and stimulate transcription
these factors have two independent domains:
- one region binds DNA
- the other stimulates transcription by interacting with other proteins such as mediator
transcriptional activators
“eukaryotes”
many different transcription factors have now been identified in eukaryotic cells. About 2000 are encoded in the human genome. They contain many distinct types of __________________
DNA-binding domains
“eukaryotes”
DNA BINDING DOMAINS
- __________: binds zinc ions and folds into loops (“fingers”) that bind DNA
________________: have zinc fingers: they regulate gene transcription in response to hormones such as estrogen and testosterone
- ___________domain: one helix makes most of the contacts with DNA, the other helices lie across the complex to stabilize the interaction
_________________: proteins are important in regulation of gene expression during embryonic development
- zinc finger domain
steroid hormone receptors
- helix-turn-helix
homeodomain
“eukaryotes”
3. ________________and ________ proteins contain DNA- binding domains formed by dimerization of two polypeptide chains.
Different members of each family can dimerize with one another-combinations can form an expanded array of factors
leucine zipper and helix-loop-helix
“eukaryotes”
activation domains stimulate transcription by two mechanisms:
- Interact with ____ and ___________
- interact with ______ to modify _________-
- mediator proteins ad general transcription factors
2. chromatin structure
“eukaryotes”
gene expression is also regulated by repressors which inhibit transcription.
In some cases they simply ____________ factors
interfere with binding of other transcription factors
“eukaryotes”
other repressors ____ for binding to specific regulatory sequences
compete with activators
“eukaryotes”
active repressors have specific domains that inhibit transcription via protein-protein interactions. These include interaction with specific activator proteins, with mediator proteins or general transcription factors, and with _______ that act by modifying chromatin structure
corepressors
transcription can also be regulated at ________-
elongation
recent studies show that many genes have molecules of RNA polymerase II that have started transcription but are stalled immediately downstream of _______
promoters
following initiation, THE POLYMERASE PAUSES WITHIN ABOUT 50 NUCLEOTIDES due to _________ including ____ (negative elongation factor) and _________.
continuation depend on another factor: P-TEFb (positive transcription-elongation factor-b)
negative regulatory factors
including NELF and DSIF
because eukaryotic DNA is packaged in _______, _____ structure is a critical aspect of gene expression
packaged in chromatin, chromatin
histones can be modified several ways- this makes them a ____ mechanism for regulating gene expression
key mechanism
many histone modifications are stably inherited when ________
cells divide
the basic unit of chromatin is the nucleosome: __________ of DNA wrapped around two molecules each of histones H2A, H2B, H3, and H4
147 bp of DNA
one molecules of histone H1 is bound to the DNA as it enter the ___________
nucleosome core particle
- chromatin can be altered by histone modifications and _____________
nucleosome rearrangments
- HISTONE ACETYLATION:
The amino-terminal tail domains of core histones are rich in _____ and can be modified by acetylation. Transcriptional activators and repressors are associated with histone ____________ and _______ respectively
Lysine
acetyltransferases (HAT)
deacetylases (HDAC)
_________ neutralizes the positive charge of lysine, relaxing chromatin structure and increasing availiability of the DNA template for transcripton
acetylation
trancriptional ____ and ____ are associated with HAT and HDAC
activators and repressors
- Histones can be modified by methylation of ______ and ______ residues, phosphorylation of _______, and additional of small peptides (ubiquitin and SUMO) to lysine residues.
these modifications occur at specific amino acid residues in the HISTONE TAIL
Lysine
arginine
serine residues
- histone modification affect gene expression by altering chromatin properties, and by providing binding sites for proteins that activates or repress transcription.
methylated H3 lysine-9 and -27 residues are binding sites for proteins that induce ________________
chromatin condensation and formation of heterochromatin
- promoters and enhancers are distinct chromatin features. They are ______, and thus accessible for binding transcription factors.
these regions can be digested by DNase (___________)
free of nucleosomes
DNase hypersensitive sites
__________________ are protein complexes that alter contacts between DNA and histones
they can reposition nucleosomes, change the conformation of nucleosomes, or eject nucleosomes from DNA
chromatin remodeling factors
following initiation of transcription, elongation is facilitated by _________ associated with the phosphorylated _______ of RNA polymerase II.
elongation factors include ____________ and _________
elongation factors
c-terminal domain
histone modifying enzymes and chromatin remodeling factors
modified histones serve as binding sites for proteins that themselves catalyze histone modifications.
histone modifications can thus regulate one another, leading to the stable patters of modified chromatin.
this provides a mechanism for _________ - transmission of information that is not the DNA sequence
epigenetic inheritance
modified histones are transferred to both progeny chromosomes where they direct similar modification of new histones- ______________________________________-
maintaining characteristic patters of modification
_______________ also controls transcription in eukaryotes.
methyl groups are added at the 5’ carbon position of cytosine (C) that precede guanines (G) (cpG dinucleotides)
this methylation is correlated with transcriptonal regression
DNA methylation
____ is a mechanism for epigenetic inheritance. Following DNA replication, an enzyme methylates CpG sequences of a daughter strand that is hydrogen-bonded to a methylated parental strand
DNA methylation
DNA methylation plays a role in _______________: the expression of some genes depends on whether they come from the mother or the father
Example: gene H19 is transcribed only from the maternal copy. It is specifically methylated during the development of male, but not female, germ cells
genomic imprinting
transcription can also be regulated by noncoding RNA molecules:
-miRNAs (20-30nucleotides) act by the RNA interference pathway to inhibit translation or induce degration of homologou mRNAs.
__________ (lncRNAs)(>200 nucleotides)
long noncoding RNAS
for complexes with proteins that ___________________ and ___________________________________, thereby regulating expression of neighboring genes
modify chromatin
recruit these complexes to their sites of transcripton
in many cases, lncRNAs repress their target genes by forming complexes with __________.
includes Xist IncRNA, which mediates X chromosome inactivation in mammals and several IncRNAs invoved in imprinting.
IncRNAs can associate with different chromatin-modifying enzymes and function as repressors or activators.
Some IncRNAs act in trans by recruiting chromatin-modifying complexes (e.g. PRC2) to distant target genes. Many other IncRNAs regulate gene expression in multiple ways
Polycomb Repressive Complex 2 (PRC2)
___________ are used immediately for protein synthesis while still being transcribed
bacterial mRNAs
__________ must be process in various ways in both prokaryotic and eukaryotic cells.
Regulation of processing provides another level of control of gene expression . Ribosomal RNAs of both prokaryotes and eukaryotes are derived from a single long ____________ molecule
Other RNAs
pre-rRNA
in prokaryotes, this is cleaved to form three rRNAs (_______, _______, _______)
16S, 23S, and 5S
eukaryotes have four rRNAs (_________, ________, ______, _____); 5S rRNA is transcribed from a separate gene
16S, 5.8S,28S,5S
tRNAs also start as long precursors (_____) in prokaryotes and eukaryotes. Processing of the ______ involves cleavage by the enzymes ___________
pre-tRNAS
5’ end of pre-tRNAS
RNase P
RNase P is a ________- an enzyme in which RNA rather than protein is responsible for catalytic activity
ribozyme
processing of the ______ involves addition of a CCA TERMINUS, the site of amino acid attachment
Bases are also modified at specific positions. About 10% of bases are modified.
3’ end of tRNAs
in eukaryotes, ____ are extensively modified __________
pre-mRNAs
before export from the nucleus
- transcription and processing are coupled.
- the C-terminal domain (CTD) of RNA polymerase II plays a key role in coordinating these process
- the 5’ end of the transcript is modified by addition of a __________________________.
the 5’ cap stabilizes the RNA, and aligns it on the ribosome during translation
7-methylguanosine cap
at the 3’ end a poly-A tail is added by ____________
polyadenylation
signals for polyadenylation include a highly conserved _______ and ____________ element
hexanucleotide (AAUAAA in mammalian cells) and a G-U rich downstream sequence element
poly-A-polymerase then adds about _____ to form the poly-A tail
200 addenines
reconition of the polyadenylation signal leads to ______,________ and _______ of mRNA
the RNA that has been synthesized downstream of hte site of poly-A addition is degraded.
introns (noncoding sequences) are removd from pre-mRNA by splicing
in mammals, most genes contain multiple introns
splicing has to be highly specific to yield functional mRNAS
leads to termination of transcription, cleave, and polyadenylation of mRNA
GENERAL
splicing proceeds in two steps:
- cleavage at the 5’ splice site (SS) and joining of the 5’ end of the intron to an A within the intron (branch point). the intron forms a loop
- cleavage at the 3’ SS and simultaneous ligation of the exons excises the intron loop
X
three sequence elements of pre-mRNAs are important: at the __________at the ___________ and within the introns at the _____________
pre-mRNAs contain similar consensus sequences at each of these points
5’ splice site
3’ splice site
branch point
splicing takes place in large complexes, called _________, which ave five types of ____________________-U1, U2, U4, U5, U6
splicesomes
small nuclear RNAs (snRNAs)
the snRNAs are complexed with 6-10 protein molecules to form ____________________
recognition of 5’ SS involves base paring between the 5’ SS consensus sequence and a complementary sequence at the 5’ end of U1 snRNA
small nuclear ribonucleoprotein particles (snRNPs)
U2 snRNP then binds to the ___________
The other snRNPs join the complex and act together to form the intron loop, and maintain the association of the 5’ 3’ exons so they can be ligated followed by excision of the intron.
this is followed by exision of the intron
branch point
________ processing (other than splicing) that can alter the protein coding sequences of mRNAs.
it involves single base modification reactions such as deamination of cytosine to uridine and adenosine to inosine
RNA editing
Editing of mRNA for apolipoprotein B, which transport lipids in the blood results in two different proteins.
- apo-B100, synthesized in the _____ by translation of unedited mRNA
- Apo-B48, synthesized in the ________ from edited mRNA in which a C has been changed to a U by deamination
liver
intestine
___________- over 90% sequences are introns, which are degraded in the nucleus after splicing. Processed mRNAs are protected by capping and polyadenylation, but the unprotected ends of introns are recognized and degraded by enzymes
intracellular levels of any RNa are determined by a balance between synthesis and degradation
rate of degration can thus control gene expression
pre-mRNA
_________________________- are very stable, in both prokaryotes and eukaryotes. accounts for high levels of these RNAs (greater than 90% of all RNAs)
rRNAs and tRNAs
mRNA
- __________________- are rapidly degraded, most half lives: 2 to 3 minutes
- __________, mRNA half-lives vary: less than 30 minutes to 20 hours.
- ____________ code for REGULATORY PROTIENS. levels of which can vary rapidly in resonse to the enviromental stimuli
- _______________ enconding structural proteins or central metabolic enzymes
- bacterial mRNAs
- Eukaryotic cells
- Short-lived mRNAs
- long half0lives mRNAs
- degration of eukaryote mRNAs:
- initated: _________________
- rapidly degraded mRNAs often contain specific AU-rich sequences near the 3’ ends-binding sites for protiens that can either stabilize them or target them for degration
shortening of the poly-A tails
- Regulation
- _____________, such as growth factors and hormones
- degradation of some mRNAs is regulated by both ______ and ___________-
extracellular signals
regulated by both siRNAs and miRNAs
