Flashcards in Transcription, RNA Processing, Gene Regulation, and Epigenetics Deck (155):
Besides the change between T and U, the RNA product should be identical to the
DNA Coding strand
The unit of prokaryotic RNA Polymerase that associates with the core enzyme to generate the holoenzyme
What are the three stages of transcription?
Initiation is dependent on what two thing?
1.) σ subunit binding RNA polymerase (making holoenzyme)
2.) Holo-RNA Polymerase binds promoter
What is the principal site for regulation of transcription?
What initiates the elongation step?
σ dissociates from RNA polymerase and the promoter
Transcript is lengthened by the addition of nucleotides to the 3' end of the RNA strand located in the active site of RNA Polymerase II
What happens during termination?
1.) RNA synthesis stops
2.) RNA transcript is released
3.) Dissociation of RNA polymerase from DNA template
What are the two sequences within the prokaryotic promoter region that are recognized by the RNA polymerase holoenzyme?
-35 sequence (TTGACA) and Pribnow box (TATAAT)
Located ~7 base pairs upstream of the start of transcription
Pribnow box (TATAAT)
What is the distance between the -35 sequence and the Pribnow box?
Can make DNA from RNA
Is translation reversible?
How many different RNA polymerases are there for
1.) Prokaryotic transcription
2.) Eukaryotic transcription
2.) Three (with a 4th that functions in mitochondrial transcription)
The structural and catalytic component of ribosomes
rRNA comprises about
80% of cellular RNA
The ribosome includes four different rRNAs that are typically designated by their sedimentation coefficients. For example, in human cells, there is
5S, 5.8S, 18S, and 28S
Functions as an "adaptor" molecule that delivers amino acids to the ribosome
-about 15% of cellular RNA
The template for protein synthesis, and is heterogeneous in size, varying according to the length of the encoded protein
-typically represents less than 5% of total RNA content
small nuclear RNAs (snRNAs) are involved in
Small nucleolar RNAs (snoRNAs) function in
microRNAs play important roles in
Regulation of gene expression
A cluster of genes encoding proteins involved in sugar utilization
What type of reaction is the polymerization that forms RNA?
Nucleophilic attack by 3' OH of growing strand on α-phosphate of incoming NTP (leaving group is pyrophosphate [PPi])
In contrast to DNA replication, RNA transcription does not require a
The core enzyme RNA polymerase is catalytically active but unable to recognize specific promoter DNA sequences until
σ subunit binds
Although they have only one core RNA polymerase, bacteria have several
What gets rid of the positive supercoils generated in the DNA by RNA polymerase during transcription?
What gets rid of the negative supercoils generated in DNA by RNA polymerase during transcription?
What is the actual site of transcription initiation?
Transcription is regulated primarily at the level of
Either recruit RNA polymerase to the promoter, or stabilize its binding to promoter DNA
Block RNA polymerases interaction with DNA
Transcription termination signals are present at the ends of genes, but function at the
What are the two classes of bacterial terminators?
Binds a specific RNA sequence as a hexameric protein and contacts RNA polymerase, signalling the polymerase to terminate transcription and dissociate from the DNA template
RNA hairpin structures generated by palindromic repeats followed by a U rich region
Rho independent terminator
In the case of rho-independent termination, a specific structure (stem and loop) forms in the RNA transcript, signalling
Binds the β subunit of bacterial RNA polymerase and inhibits initiation
Rifampicin is an effective antibiotic for treatment of certain
Bacterial infections (ex: tuberculosis)
Binds the DNA template, intercalating between neighboring base pairs
The DNA structure that binds actinomycin is conserved between prokaryotes and eukaryotes. As a result, actinomycin serves as an effective therapeutic for treatment of some
Actinomycin binds the
DNA phenoxazone ring
The core promoter region of eukaryotic class II genes typically (but not always) contains a
-located about 25 bp upstream of the transcription start site (+1)
The “nucleation site” for assembly of a transcription complex that includes a set of general transcription factors (GTFs) and RNA polymerase II and is functionally comparable to the –10 and –35 regions of a bacterial promoter.
Transcriptional stimulatory sequences are generally located upstream of the core promoter in eukaryotes and include
Promoter proximal elements and enhancer sequences
Promoter proximal elements are usually located within 120 bp of the transcription start site and serve as binding sites for transcriptional activators of
-Ex: enzymes involved in metabolism
Ubiquitously expressed genes ("housekeeping genes")
The most well characterized activator that functions at a proximal promoter is called
Found at variable distances from the core promoter and are DNA sequences that bind specific transcriptional activators
Binding sites for transcriptional activators that either make direct contact components of the RNA polymerase II transcriptional machinery, or recruit chromatin remodeling complexes to the regulatory promoter regions
What do RNA Polymerases I, II, and III transcribe?
RNA Polymerase I = rRNA
RNA Polymerase II = mRNA
RNA Polymerase III = 5s component of ribosome, tRNA, and certain snRNAs
Eukaryotic RNA polymerases do not recognize Specific DNA sequences on their own. Instead, promoter recognition requires
General transcription factors (GTFs)
A general transcription factor which includes TATA binding protein
Required to position RNA Polymerase II at the promoter
Structurally similar to prokaryotic RNA polymerase
Eukaryotic RNA polymerase II
As a subunit of the larger TFIID complex, binds the TATA element and confers a sharp bend in the promoter DNA.
TATA binding protein (TBP)
Explain the process of RNA polymerase II binding
TBP (as TFIID) binds the TATA box, enabling TFIIB to bind, which enables RNA polymerase II and TFIIF to bind
Once RNA polymerase II is bound to DNA, enable RNA polymerase to start transcription
TFIIE and TFIIH
Stimulate transcription by binding GTFs, either to recruit or stabilize their binding to the core promoter
There are two notable features of eukaryotic RNA pol II transcription that do not occur either in bacteria or by RNA pol I or RNA pol III. These are
Addition of 5' cap and 3'-poly(A) tail
A 7-methylguanosine “CAP” is covalently linked to the 5’-end of mRNA by a unique 5’ → 5’ triphosphate linkage. The CAP is not encoded by the DNA template. Instead, CAP is added to the mRNA shortly after
Consistent with it being a unique feature of mRNA, the CAP is recognized by components of the translational machinery, thereby facilitating
Transcription termination by RNA polymerase II involves the addition of a
Termination is specified by distinct sequences in the DNA template and involves which two enzymatic reactions?
1.) Endonucleolytic cleavage of nascent transcript
2.) Addition of poly(A) tail
The poly(A) tail is not encoded in the DNA template and is a unique feature of eukaryotic RNA pol II transcription. The poly(A) tail is involved in
mRNA stabilization, transport of mRNA from nucleus to cytoplasm, and efficient translation
Unlike DNA replication, which is semi-conservative, trancription is
In eukaryotic transcription, the core and regulatory elements can be far apart, but in prokaryotes, they must be
In prokaryotes, translation of the mRNA begins
Prior to transcription termination
In ekaryotes, transcription takes place in the nucleus and translation in the
In prokaryotes, The 5' end in the DNA sequence of a gene is directly proportional to the
N-terminus of the protein
In most eukaryotic genes, the coding information is
Discontinuous (introns and exons)
Nucleic acid sequences that are transcribed and retained in the corresponding mature mRNA
Nucleic acid sequences that are transcribed but spliced from the primary transcript to yield the mature mRNA
A gene that is divided into 3 exons and 2 introns
-The ATG start codon is found in exon 1
-The TAA stop codon is contained in exon 3
Why must the translation start and stop codons be within exons?
Because introns are spliced out either co- or post-transcriptionally, but PRIOR to translation
When the intron sequences are removed, the adjacent exons are
pre-mRNA splicing is very important because a single nucleotide error in the splice point would have what consequence?
The reading frame would be shifted, resulting in an entirely different amino acid sequence
The base sequence of an intron
1.) Begins with?
2.) Ends with?
-these sequences are invarient (never change)
A conserved sequence found 20-50 nucleotides from the 3' end of the intron
The "branch" site
Intron sequences other than the 5' and 3' splice sites, and the "branch site", are unimportant in determining
Location of splicing
The length of an intron varies from
Less than 100 nucleotides to several thousand nucleotides
Intron splicing occurs by two
The first transesterification reaction involves
Cleaving the phosphodiester bond between exon 1 and intron 1, and using the 2' OH of an adenylate residue at the branch site to form a phosphodiester bond with the 5' end of the intron
In the second transesterification, the 3' OH of exon 1 cleaves the phosphodiester bond between the intron and exon 2. As a result,
Exon 1 and 2 are now bound together, and the intron is released as a "lariat" structure
An assembly of ribonucleoprotein particles (SNURPs) that recognize the 5' splice sit, the 3' splice site, and the branch site
-Catalyzes the splicing reaction
The spliceosome assembles de novo from its constituent SNURPs on the precursor RNA in a process that requires
Required for the spliceosome assembly, but not for either of the ensuing transesterification reactions
Which spliceosome protein(s) bind the
1.) GU 5' splice site
2.) Branch site A
Approximately 15% of mutations that cause genetic disease affect
A group of hereditary anemias characterized by defective synthesis of hemoglobin
One source of Thalassemias is due to point mutations that occur in either of the two β-globin gene
Point mutations in β-globin gene introns create
-results in either shorter or longer forms of abnormal β-globin
What are three DNA sequence elements in eukaryotic transcription?
1.) Enhancers (>250)
2.) Proximal promoter (CpG: -120 to -60)
3.) Core promoter (TATA: -40 to +40)
In eukaryotes, contains the transcription start site (+1), the TATA box (-25), and down stream promoter element (DPE: +30)
The proximal promoter is the binding site for certain
What are the core promoter elements of RNA polymerase II?
2.) Down stream promoter element (DPE)
3.) TATA Box
4.) TFIIB Recognition element
Nucleates the assembly of the transcription preinitiation complex
-TBP + 14 TBP-associated factors (TAFs)
How does TFIID work?
TBP binds TATA box, TAF1 and TAF2 bind initiator, and TAF 6 and TAF 9 bind the DPE
Binds tightly to RNA Polymerase II and inhibits the elongation stage of transcription
-Eukaryotic version of Rifampicin
Can produce multiple, related proteins from a single gene
What is the Donor splice site?
5' end of intron
What is the Acceptor splice site?
3' end of intron
Many hormones (e.g. epinephrine, insulin, epidermal growth factor) activate transcription by activating
Directly stimulate gene transcription as hormone-receptor complexes
Humans contain two copies (one from the mother and one from the father) of each
Soluble proteins that function as transcriptional activators in response to hormone binding
Steroid hormone receptors
In come cases, hormone binding exposes a nuclear localization signal, allowing the receptor-hormone complex to enter the nucleus, where it binds
Promoter DNA (in a sequence specific manner)
In other cases, the hormone receptor is already located in the nucleus. In this case, hormone binding does one of what two things?
1.) Stimulates receptor-DNA binding
2.) induces conformational change in the receptor-DNA complex
Regardless of the specific mechanism, the DNA-hormone-receptor complex then interacts with a "coactivator" complex to stimulate
RNA Polymerase II transcriptional machinery
Steroid hormones receptors, like other transcriptional activators, consist of discrete functional domains. What are they?
DNA-bining domain, hormone binding domain, dimerization domain, and activation domain
Bind hormone receptors and stimulate receptor activity (gene expression)
Ex: Anabolic steroids
Bind hormone receptors and block receptor activity (repress gene expression)
Recognizes specific enhancer elements
DNA binding domain
Upon hormone binding, Steroid receptors typically undergo a conformational change to create a transcriptional activation domain that
Binds a "coactivator" complex
These DNA sequences are enhancers for steroid responsive genes
-bind hormone receptor complexes, resulting in stimulation of gene expression
Steroid response elements (SREs)
Typically palindromic, and are related to one another by subtle, but important sequence differences
Steroid hormone-receptor complexes are potent activators of transcription. The mechanism of activation involves
Binding of the hormone-receptor complex to the SRE (cognate regulatory element) and subsequent binding to a transcriptional coactivator that in turn stimulates transcription
Some co-activators interact with both activators and the core transcriptional machinery (RNA Poly II and the GTFs), where they essentially
Bridge the two components
Other coactivators do not interact with the core machinery, but instead target
Chromatin (either displacing or modifying nucleosomes)
Normally inhibit transcription
Some work by recruiting or stabilizing RNA polymerase II/GTP to the core promoter, and other "clear a path" for RNA polymerase II by altering the DNA template
DNA in eukaryotic chromosomes is not naked, but is tightly associated with proteins to form
Steroid hormone receptors ultimately activate gene expression by altering
-Allows RNA polymerase II to bind the promoter
In summary, a sequence specific enhancer element (DNA) binds the hormone receptor-hormone complex (transcriptional activator), which in turns binds a coactivator complex (chromatin modifier) to
Displace or disrupt nucleosomes
Coactivators include two classes of chromatin modifiers. What are the two classes?
1.) Coactivators that catalyze covalent modifications to histones
2.) ATP-dependent remodeling complexes
The most well characterized covalent histone modification is acetylation of histone lysine residues in the N-terminal tails of histones H3 and H4, catalyzed by
Histone acetyltransferases (HATs)
Histone modifications occur primarily at the
N-terminal tails of histones
These complexes do not catalyze covalent histone modification, but instead use the energy of ATP hydrolysis to remodel nucleosomes, thereby allowing other transcription factors to bind DNA and ultimately promote gene transcription.
ATP-dependent chromatin remodeling complexes (coactivators)
Steroid hormones include estrogens, androgens, glucocorticoids and mineralocorticoids. These cholesterol derivatives, each slightly different in structure, bind specific hormone receptors to
Activate different sets of genes
Target the oviduct to stimulate expression of genes whose encoded proteins participate in the ovarian cycle
Target the liver (and other tissues) to promote expression of genes required for gluconeogenesis, glycogen synthesis, degredation of fat, and inhibition of the inflammatory response
A DNA virus that causes breast epithelial cell carcinoma. It's genome includes a DNA sequence that is identical to the estrogen enhancer
Mouse mammary tumor virus (MMTV)
In MMTV, the estrogen/estrogen receptor complex binds the MMTV enhancer and stimulates wnt-1 gene expression, which results in
Uncontrolled cell proliferation
Anti-cancer drug that binds the estrogen receptor and prevents coactivator binding
Despite being a "general transcription factor" required for the expression of all protein genes, TFIIH also plys a very important role in the
Coupling of transcription with DNA damage repair
Altered forms of TFIIH have been identified in what three diseases?
-These diseases are not due to defects in transcription initiation, but rather to defects in DNA repair
1.) Xeroderma pigmentosum (XP)
2.) Trichothio-dystrophy (TDD)
3.) Cockayne syndrome (CS)
Will move 3' to 5' on the DNA template to generate a new 5' to 3' strand
The sequences indicating the -35 and TATAAT box are not exact, but rather consensus sequence. All six of the consensus nucleotides are never actually seen in the same gene because
Sigma would bind too tightly
The σ subunit binds the -35 and TATAAT sequences
Which sigma unit recognizes E coli promoters?
A double stranded DNA sequence that the σ subunit binds to
What are the 5 catalytic subunits of RNA polymerase II?
2α, β, β', and ω
Found in the crab claw of RNA polymerase II and help make up the active site
Two magnesium ions
The core promoter spans about
80 base pairs
Not a promoter in the sense that it does not bind
RNA polymerase. Binds proteins that are regulatory proteins for "housekeeping" genes (genes encoding proteins that every cell needs to have)
Core promoter, proximal promoter, and enhancers are all
Which GTF binds to -35?
The only place a 5' to 5' triphosphate linkage occurs is on the
-prevents degredation of mRNA and facilitates the initiation of translation
The code to terminate transcription is in the
DNA template and has the consensus sequence
Transcription factors that bind to double stranded DNA
What is a common structural motif in transcription factors?
Has 1.74 turns of double stranded DNA and about 147 bps
-an impediment to RNA polymerase
Rely on estrogen-mediated pathways for cell proliferation
-Why tomoxifin is a good anti-cancer