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83 Cards in this Set

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What is the definition of a gene?
The entire DNA sequence tt's necessary for the production of one functional RNA or protein molecule
What sequences does the gene have in regards to transcription?
1. The coding region, or "transcription unit"
2. The transcription control regions, wc often lay outside the coding region
There are several differences b/w Prokaryotic transcription and Eukaryotic. Prokaryots are polysystronic while Eukaryotes are monocystronic. What does ts mean?
Prokaryotic RNA may code for more than one protein. Eukaryotic RNA only codes for one protein.
The coding sequence is different in Eukaryotes than Prokaryotes; Prokaryotes lack a certain type of coding region that Eukaryotes have, wt is it?
Eukaryotes hv segments of non-coding regions b/w the code regions; ts allows for more tn one combination of info being spliced tgtr. Prokaryots are "simple", ty tr coding regions are not broken up by the non-coding sequences.
What is the difference b/w the location of related genes in the genome in Eukaryotes vs. Prokaryotes?
Prokaryotes genes/transcription units are densely packed in the genome, w/neighbors often related and showing little non-functional DNA b/w the units. Related genes in Eukaryotic genome can be widely dispersed often w/extensive non-functional & repetitive DNA in b/w
Another difference b/w Prok. and Euk. is b/w the controlling of promoter/operators. Wt is the diff?
Prok. transcription units r controlled via a proximal "promoter/operator" combo. Euk. units tend to hv multiple proximal and distal control elements
What happens in initiation of transcription?
Polymerase enzyme binds to promoter sequence and forms the 1st phospodiester bonds.
What step and where is the main site of transcriptional regulation and thus control of gene expression in general
Initiation; the promoter region is the main site of transcriptional regulation and thus control of gene expression in general
What protein is responsible for the addition of nucleotides to the growing chain (elongation)?
RNA Polymerase
There is only one RNA polymerase in prokaryotes. Describe its structure.
-Its "core" polymerase is composed of 5 subunits: 2 ß and ß' wc can grip the DNA and house basic catalytic site, 2 α subunits and ω
Describe the interactions of the subunits during transcriptions/initiation
- The α units interact w/regulatory proteins to "activate" transcription by the ß units to wc ty bind.
- The ω unit assembles the others into a complex.
- σ attaches, forming the RNA polymerase "Holoenzyme", wc is able to initiate translation; it is the σ tt recognizes the promoter
There are 3 different classes of eukaryotic RNA polymerases, based on their structure and functions. What are the 3 classes?
RNA Polymerase I, RNA Polymerase II, RNA Polymerase III
Where are each of the RNA polymerases located?
RNA Polymerase 1 = only in nucleolus
RNA Polymerase 2 = General nucleoplasm
RNA Polymerase III = specific "speckled" sites distributed trghout the nucleus
What does RNA Polymerase I do?
- Synthesizes a pre-rRNA tt's processed into ribosomal rRNA subunits
- Synthesizes 50% of the RNA and 75% of the RNA transported to the cytoplasm
What does RNA Polymerase II do?
- Initiates transcription fm promoters for all protein coding genes as well as fm the promoter for the sm nuclear RNAs (snRNAs) involved in splicing
- Transcribes 40% of the nuclear RNAs, wc yields 10% of cytoplasmic RNA
What does RNA Polymerase III do?
- Transcribes genes for many sm RNAs including the 5S rRNA, all tRNAs and several sm stable cellular RNAs (scRNAs)
- Accounts for 10% of RNA synthesized and 15% of the RNA located in the cytoplasm
What are the degrees of affectedness of each RNA polymerase to the poison α amanitin?
Polymerase I = unaffected
Polymerase II = nearly completely inhibited by
Polymerase III = intermediate sensitivity
What is the general structure for the Eukaryotic RNA polymerases?
They contain subunits that are "orthologs (structurally or functionally equivelent) to the prokaryotic ß,ß',α,ω subunits. Ty hv 5 eukaryotic specific subunits + a series of subunits unique to each form. A series of general and specific "transcription Factors" take the place of the σ subunit in order to control the more complex process of eukaryotic transcription
Prokaryotes: In ____ it has been found tt the core enzyme can copy any sequence of DNA. In ____, however, it will only initiate at the promoter
In Vitro. In vivo.
Prokaryotes: The promotor contains DNA sequence elements that do 2 main things, wt r ty?
1. direct the tight/strong binding of the Core Polymerase and its accessory, helper proteins to the DNA
2. Contains info tt determines the direction of transcription, the strand of DNA to be copied, & precise starting point of RNA synthesis
There are two important sequences of DNA upstream of the promotor in Prokaryotes, wt r ty?
Both are 6 nucleotides each
1. -10 aka Pribnow box (TATAAT)
2. -35 box (TTGACA
What events occur during initiation? (Prok)
- The σ recognizes and tightly binds to the -10 and -35 boxes (the holoenzyme can weakly bind DNA). This locks the core enzyme inplace over the true start site.
-Polymerase unwinds ~17 b.p.s surrounding the "start" site, creating the transcription bubble
-The "sense" strand serves as template. THe nonsense strand will hv identical sequence to the new strand
- Formation os the 1st phosphodiester bond b/w a base pair is considered the "initiation" event. The intiation phase" isn't complete til ~10 nucleotides added.
Once Polymerase binds to the DNA, how many base pairs will it cover?
~70. Wn at initiation, ts includes 20 bases downstream of the start and 50 upstream.
What are the "steps" the polymerase performs to form the bonds b/w the nucleotides?
Polymerase looks at the H bonding pattern of the template to orient the 1st 2 nucleotides side by side. Once the proper H-bond pattern is recognized, it catalyzes the formation of a phosphodiester bond
Prok: What happens to shift the transcription from "initiation phase" to "elongation phase"?
Wn polymerase transcribes it induces a conformational change in itself. Ts displaces the σ & causes the polymerase to enhance its grip on the DNA and enter the "elongation" phase; there are several steps involved.
What are the 4 steps involved w/the start of elongation? (I think ts is actually the process of transcription in general)
1. Binding a free nucleotide
2. H-bonds made tt changes conformation of enzyme, locking nucleotide in its active site
3. Enzyme catalyzes the attachment of 5'α phosphate of free nucleotide to exposed 3'OH of last nucleotide. Ts displaces pyrophosphate tt can be hydrolyzed later for release of energy
4. Formation of phosphodiester bond induces further conformational change in enzyme tt moves it forward 1 base on template
Prok: Can polymerase unzip the DNA? Can it separate the DNA template fm new RNA strand as it moves along in transcription?
Yes it can both unzip the DNA helix and separate the RNA product fm DNA template.
Does RNA polymerase have an editing function? (Prok)
Not technically; it is able to back up and remove an added base, but it doesn't hv a true "editing" function like DNA polymerase, thus RNA synthesis is more error prone
In bacteria, tr are two forms of termination what are ty called?
Type I and Type II
What is Type I termination in bacteria?
-Simplest and most common
- Termination signal consists of ~20 GC rich nucleotides tt r inverted repeats followed by 4 or more A residues (AAAA)-The inverted repeats form a stem-loop structure wn transcribed tt will bind to rear of polymerase. Continued forward motion will "pry the RNA fm the DNA at the weakly H bonded A-U stretch
What is Type II termination in bacteria?
A protein factor called rho binds to recognition site near end of transcribed RNA. It uses ATP to unwind the RNA fm the DNA template, preventing further synthesis
How many RNA polymerases are there in Eukaryotes and wc is emphasized in the notes?
3 polymerases, RNA polymerase II, wc produces mRNA templates for protein synthesis, is emphasized.
Euk: What 2 primary sequences comprise the promoter region?
1. TATA box
2. Inr sequence
What is the TATA box comprised of and located?
It's a sequence of 6 positioned 25-35 upstream fm start site.
Which is more important; TATA box or Inr sequence? Why?
TATA box: It serves as the primary binding site for the plymerase and its "initiation factors" and thus determines the precise starting nucleotide.
In prokayotes, σ is used to recognize and bind polymerase to the promoter, what is used it Eukaryotes?
Numerous General Transcription Factors for RNA polymerase(gTFIIs)
In prokaryotes, σ can only recognize and bind to promoter. In Eukaryotes, the transcription factors do this as well as what else?
They are involved not only in promoter recognition but in transcription as well
How many Transcription Factors are there in Eukaryotes and wt r tr names?
2. TFIIA (Activator)
3. TFIIB (Binder)
4. TFIIF (Ferry)
5. TFIIE (Enhancer)
6. TFIIH (Helicase)
How many functional activites does TFIID have, wt r ty? (Euk)
Has 2 functional activites: the TATA Binding Protein (TBP) & the TATA Associated Factors (TAFs)
What do TAFs do? (Euk)
- Ty're regualtory subunits
- Ty couple the activity of "Specific Transcription Factors" to the activation of the polymerase.
What does the TBP do? (Euk)
Recognizes and binds to the TATA Box. Ts distorts the DNA making it easier to unwind and changes the conformaiton of TBP, the gTFIIs and ultimately the polymerase promoting the initiation process
What does TFIIA (Activator) do? (Euk)
Binds to the TATA localized TBP preventing inhibitors fm interfering w/process thus "activating" transcription
What does TFIIB (Binder) do? (Euk)
Binds to the TBP and to incoming polymerase. It determines the direction of transcription and the position of the 1st base to be transcribed
What does TFIIF (Ferry) do? (Euk)
Routinely found in association w/free polymerase. It carries (or ferries) the polymerase to the initiation complex at the promoter region by coupling w/IIB
What does TFIIE (Enhancer) do? (Euk)
Found in a complex w/IIH. It carries IIH into initiation complex and tn enhances its enzyme activities.
What does TFIIH (Helicase) do? (Euk)
1. Unwinds the DNA creating a transcription bubble
2. Acts as a kinase: Phosphorylates the tail, called Carboxy Terminal Domain (CTD), of the polymerase. This allows the polymerase to unattach itself fm the gTFIIs and begin elongation
Is Elongation similiar in prokaryotes and eukaryotes?
What happens once the CTD is phosphorylated and the initiation factors are released?
Polymerase will bind RNA processing enzymes, initiating the mRNA processing sequence (Capping, Splicing, Tailing)
Describe capping in Eukaryotes
- occurs after 30 nucleotides
- Involves addition of an inverted Guanine nucleotide to the 1st nuclecotide in new chain. (in a 5'-5' link)
- Modification of guanine and sugars of the 1st 2 bases by addition of protective methyl groups
What does capping do (ie wt's its purpose)?
1. Mark beginning of the RNA
2. Protect fm degradation during processing since the 5'--5' linkage is not recognized by most nucleases
3. Provide a special binding site for RNA transport and protein synthesis factors
Which is spliced introns or exons?
Introns are spliced
How is an intron recognized?
Each intron is marked by identifying nucleotide sequences; each intron begins w/GU and ends w/AG and has an invariant A base ~20 residues fm 3'end
What is the name of the complex that carries out splicing?
What are the steps in splicing? (Eukaryotes)
Look at notes; section 11, page 6
What happens in the termination phase of RNA processing?
Endonucleolytic cleavage results in 3' end being modified by the addition of a series of non-coded A bases (polyadenylation signal).
What is the polyadenylation signal? (Euk)
Can be either of 2:
1. AAUAAA sequence 10-35 nucleotides upstream fm the polyA cleavage site
2. GU rich region downstream
What is the current proposed model for the process of adding a Tail to RNA?
See notes, section 11, pg 7
What is the primary function of gene control in prokaryotes (and many unicellular eukaryotes)?
To adjust the enzymatic machinery of cell to its immediate nutritional and physical environment
What is an example of an operon that acts as a control of transcription in bacteria? Wt's it consist of?
Lactose Operon; consists of an "i gene"
What does the "i gene" of Lactose Operon do?
constitutively produces a repressor protein & 3 structural genes coding for the proteins ß-galactosidase, permease, and transacetylase.
What is an operon?
A cluster of genes whose expressions are regulated tgtr by operato-regulator protein interactions
What is meant by "ordinary condition" for a cell, in regards to lactose? (bacteria)
The bacterial growth is on a medium of glucose, with lactose being absent
What happens under ordinary conditions in regards to the control of Lactose? (bacteria)
- the repressor protein will be bound to the operator
- Ts blocks the access of the polymerase from the promoter and thus inhibites gene activity
- When lactose is present, however, it binds to the repressor allostericlally adjusting its conformation so it can't bind to the DNA.
-This allows transcription of the operon
What is the primary function of gene control in Eukaryotes?
The gene regulation is directed toward the execution of a precisely positioned and timed developmental/diffrentiation program tt results in organ systems able to maintain the proper internal environment for the cells.
In prokaryotes, the repressor acted to inhibit the production of an operon while the operator acted to transcribe the operon. In eukaryotes, a similiar "system" is in place. What is it?
Control results fm the interaction of "Cis Elements" and "Trans Factors"
What are "Cis Elements" and "Trans Factors"? What are their bacterial equivilents?
1. Cis: DNA sequences associated directly w/the transcription unit to be controlled
2. Trans: proteins tt translocate thrgh the nucleoplasm to bind w/Cis
What are the 2 categories of Cis Regulatory Elements in the DNA surrounding eukaryotic transcription units?
1. "Promoter Proximal"
2. "Enhancers"
What are "Promoter Proximal" Cis Regulatory Elements responsible for in Euk?
- Moderate stimulation of the controlled gene-may act in cooperatio w/others
- can be general gene activators (is found 5' to a large variety of functionally unrelated genes)
Where and how many bps long are Promoter Proximal C.R.E?
Located 200bp fm start site.
~8-10bps long
Can Promoter Proximal C.R.E be both gene or gene class specific?
Yes. Ty can be found 5' to 1 or a group of functionally related genes, for example, all ts genes needed for the differentiation of a specific cell type
What are "Enhancers" of Cis Regulatory Elements responsible for?
-Greatly stimulate transcription (100X or more)fm the gene ty control
-Ty're effective whtr ty are arranged "forward", "backward", 5', 3', or even w/in gene ty control
Where are Enhancers of CRE usually found and how long are ty?
-Usually found at a great distance (100's to 1000's of bp) fm gene controlled
- 100-200bps long and contain 1 or more eight nucleotide "octamer" sequences tt bind regulatory proteins.
What could happen if Cis Regulatory Elements are displaced, either naturally or artificially to another part of the genome?
Ty can substantially activate any gene into wc ty come into contact
What is another name for Trans Factors? Euk
Activators/Repressors, or more generally as (Specific) Transcription Factors
Trans Factors/sTF have a two domain structure, what are the two domains? Euk
1. DNA binding Domain
2. Activation Domain
What does the DNA binding Domain of Trans Factors do? Euk
-Specifically recognizes a sequence of DNA
-Recognition occurs via groups of H-bond patterns exposed in major or minor groove of DNA
What does the Activation Domain of Trans Factors do? Euk
- Relatively diverse
- Work by interacting either w/"chromatin modifying enzymes" to expose DNA regions or by interacting w/TAFs of gTFIID in order to direct the TBP to TATA sequence of the promoter and therby recruiting RNA polymerase to the gene of interest
What enables or requires some, but not all, transcription factors to act w/partners for full functionality?
The fact tt ty have a dimerization domain
There are 4 different patterns of control in eukaryotes, what are they?
1. Gene Groups
2. Networks
3. Combinanatorial Control
4. Heterogeneity of sites/factors
What is gene control of Gene Groups?
- a lg group of genes hv the same Cis element in tr 5' flanking region.
- Ts allows tm to be controlled as a unit by a single TF.
What is gene control of Networks?
-1 of the genes responding to a particular TF is itself another TF tt binds to a different Cis element
-Such networks can sequentially activate a series of genes during the developmental stages of an organ
What is gene control of Combinatorial Control?
-Ts is the standard situation for most Eukaryotic genes.
-Each gene has multiple Cis elements responding to distinct TFs
-Cooperative interaction allows not only off/on induction, but also regulation over a wide range of activities
What is gene control of Heterogeneity of sites/factors?
-Many Cis elements are made of 2 or more sequence elements.
-Ts s.e. may be inverted repeats, variations on a family of sequences, or unrelated sequences
-Ts can be combined w/tendency of many TFs to dimerize either as homo or heterodimers to generate multiple patterns of control for an individ gene depending upon the complement of transcription factor components present at a given time in a given cell