Module B Flashcards
B1- Template vs nascent polymer
Template: structure that allows molecules to line up in specific order to create macromolecule
Nascent: newly formed
B1- coding (non-template) strand
The strand complementary to the template strand. The transcripted RNA will look like this strand, except the thymine will be replaced
B1- Active site
—- where EXACTLY is the active site?
Insertion and post-insertion site, and is located in the palm of DNAP
active site consists of a binding site and a catalytic site
Binding site binds and orientates substrates.
The catalytic site reduces the activation energy.
B1- Mechanism/ pathway/ reaction steps
Trancription factors and DNAP comes together initiate replication.
B1- binding vs dissociation
Binding: something attaching onto something else
Dissociation: breaking apart into smaller parts
B1- chemical reaction vs conformational change
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Chemical reaction: forming/breaking bonds
Conformational: rearrangement of something without changing its molecular structure
B1- initiation
Start of replication and translation
Translation starts with all the subunits and RNAP attaching to the promoter on the mRNA
7 things required for initiation
1. 30s subunit 2.mRNA 3.tRNA-fMet 4. IF(initiation factors) 1,2,3 5. GTP 6. 50s subunit 7. Mg2+
B1- origin of replication
Origin starts at region rich in A=T since it has only 2 hydrogen bonds thus easier to open.
It will proceed bi-directionally
B1- promoter
RNAP will bind to promoter sequences to initiate transcription
Promoter is a sequence of genes that direct transcription of adjacent genes. The promoter is not transcribed.
B1- ribosome-binding/shine-Dalgarno sequence
a group of 4-9 purines (AG) residues 8-13 bp upstream of +1 nucleotide
which binds to the 16S rRNA in the ribosome
B1- primer
DNA polymerase needs a primer to build on, thus, it needs primase to build a short primer, which it will then build on.
Primer is made of RNA
B1- positive supercoils vs negative supercoils
positive supercoils: overwound DNA coils are located downstream (ahead) of the transcription bubble
negative supercoils are underwound DNA coils that are located behind the transcription bubble
B1- initiating tRNA
The first amino acid is fMet- bonded to tRNAf(fMet) matching the 5’AUG guided by the Shine-Dalgarno sequence
B1- replication fork
DNA replication
Helicase unzips the DNA making a replication fork
there are two replication forks since replication is bi-directional
B1- elongation-transcription RNA
the process after initiation, building of RNA
B1- insertion site vs postinsertion site (DNAP)
insertion site: incoming nucleotide is placed here
postinsertion site: after the phosphodiester is formed, the newly placed nucleotide is shifted here.
B1- Insertion site vs postinsertion site (RNAP)
? same?
B1 -A site vs P site- ribosome
Both the 50s and the 30s contribute to the characteristics of the a and p site
E site is mostly determined by the 50s
aminoacyl site: the site where an aminoacyl group attached to a tRNA
peptidyl site: the tRNA will be moved here once is no longer an aminoacyl
B1- translocation
the final step of the elongation cycle
the ribosome moves a codon from the 3’ end of the mRNA(ribosome reads from 5-3)
this causes the dipeptidyl-tRNA to shift from the A site to the P site. Also forces the P site to exit to E site.
Movement requires EF-G (translocase) and energy from GTP
B1- elongation rate
movement of replication fork is about 50 nucleotides/second in eukaryotes
and 250-1000/s in prokaryotes
for DNAP III (3)
Elongation by RNAP is e. coli is about 50-90 nucleotides/second
B1- processivity
average number of nucleotides it can add without dissociating from the substrate
DNAP III= >500,000
DNAP I=3-200
Because DNAP replaces the RNA primers it doesn’t need to transcribe for long
DNAP II= 1,500
B1- termination
termination of replication can be rho-dependent or rho-independent
Rho-independent is hairpin loop
rho-dependent (rho helicase) requires a CA-rich region (rut). The RNAP will stop at the termination site, and the rho helicase will catch up and separate the DNA and the RNA
B2-promoter -10 vs -35 vs UP region
UP region is approx. between -40 and -60, and it strongly stimulates transcription but not all promoters contain them. Is AT rich and the alpha subunit binds here.
-10 and -35 are regions in which the sigma factor (70) attach to, in order for transcription to occur
B2- consensus sequence
Most frequent residue for each position in a sequence
- it is a consensus among promoters
eg. consensus for -10 is 5’TATAAT’3
the closer the promoter is to the consensus, the more effective it is
B2- numbering convention (upstream vs downstream)
The first nucleotide transcribed is the +1 nucleotide
Upstream: the untranscribed stuff and is expressed as a negative number. The greater the number, the farther away it is from the +1 nucleotide
Downstream: opposite of upstream
B2- cistron, polycistronic
combinatorial control
polycistronic-many genes on a single transcript
eg. lac operon promoter causes all three genes to be transcribed on to
Cistron- a unit of DNA/RNA that corresponds to a single gene
B2- operon
unit of genetic expression that consists of 1 or more regulated genes, its operator, and promoter sequences.
B2- core subunit vs sigma subunit
5 Core subunits for RNAP in E.coli
1 sigma factor- number denotes the size(molecular weight)
the sigma factor binds to the core and directs it to the binding site
**RNAP II in eukaryotes contains 12 subunits
B2- holoenzyme
6 subunits of RNAP make up the holoenzyme
Holoenzyme: catalytically enzyme, an enzyme with all subunits, phosphate groups, and cofactors
B2- Primary Sigma subunit (sigma 70)
In normal conditions, the use of this subunit is predominant. However, if the cell receives an insult (eg. heat) then it may use other subunits like sigma 32 to change cell physiology to adapt to the enviroment.
B2- architectural regulators
In eukaryotes, sometimes activator and promoter sites are far apart. Proteins called architectural regulators help loop the DNA to bring the two sites closer.
B2- basal expression
The amount of expression determined solely by the promoter
no repressor and no promoter
B2- closed vs open complex
Open complex: During initiation of transcription, the bound DNA in the -10 region is partually unwound but still intact.
Closed: the bound DNA is intact.
once transcription is initiated, the complex will convert to the elongation form
B2- DNA unwinding
The unwinding of DNA is considered helicase activity since helicase is the enzyme that unwinds DNA in replication.
In transcription, the unwinding is done by THIIF at the inr sequence to form an open complex.
B2- Elongation complex
The DNA in opened and the CTD has been phosphorylated by the CK9 (though kinase activity).
B2- promoter clearance
The step before elongation, it is the process of the complex moving away from the promoter
B2- elongation factor (NusA)
proteins required for the elongation step of translation are called elongation factors.
In bacteria this consists of: EF-Tu, EF-Ts, and EF-G.
NusA replaces the sigma factor subunit once the subunit leaves. NusA prevents premature termination and also speeds up transcription of some (BOXA)
B1-3 RNAP I
It only makes one type of RNA
-pre-ribosomal RNA
and differ greatly from one species to another
B1-3 pre-ribosomal RNA (pre-rRNA)
pre-rRNA cannot be used until it has been spliced, and after splicing it becomes an rRNA (ribosome)
B1-3 Alpha-amanitin sesitivity
Blocks RNAP II and in high conc. it will also block RNAP III. However, this will affect only eukaryotes since bacteria use bacterial RNAP. Also the mushroom’s own RNAP II is not blocked.
B1-3 RNAP II
It makes the most of the mRNA in eukaryotes. It creates the mRNA templates for proteins
B1-3 general transcription factor TFII
Factors with the label TFII are highly conserved across eukaryotes (similar)
These are very important to forming the initiation complex
B1-3 TATA box/ Initiator (inr)
around -30 of the inr sequence, composed of a bunch of TATA
B1-3 TATA binding protein (TBP)
TBP binds to the TATA box, if the promoter has no TATA box then it will arrive as a complex called TFIID
B1-3 RBP1 C-terminal domain (CTD)
repeats of an amino acid code of YSPTSPS
It is separate from the main body of the enzyme by a linker sequence.
Also the CTD helps to up the methylated cap at the 5’ end of the mRNA and it coordinates interactions between complexes in post-transcription (splicing)
B1-3 phosphorylation/ kinase
Phosphorylation of the CTD is required for elongation complex to form since it coordinates interactions between complexes in post-transcription (splicing)
B1-3 RNAP III
produces tRNA and some special RNA’s
and has a highly specific promoter sequence
B1-3 recruitment
Addition of a transcription factor into the complex.
B1-3 per-initiation (closed) complex vs initiation complex (open) complex
Once the all the parts (THIIF and THIIE are the last ones) attach you form a closed complex. Then the THIIF will promote unwinding of the DNA at the start site.
B1-3 elongation complex (elongation factor) vs termination complex (termination factor)
Elongation complex: the complex after promoter clearance
Termination: after CTD dephosphorylate, and elongation factors dissociate as well as termination factors
(B1-4) expression-1156
transcription of a gene. If the gene is being transcribed than it is being expressed.
(B1-4) housekeeping genes-1156
Genes that are generally expressed constantly.
eg. genes that produce tRNA or other central metabolic pathway
(B1-4) constitutive vs. regulated-1156
constitutive: unvarying expression
regulated: expression responds to molecular signals
(B1-4) basal expression rate
This is the rate of transcription determined SOLEY by the promoter sequence.
The promoter will determine the affinity for RNAP and its transcription factors
(B1-4) inducible / induction vs. repressible / repression-
Inducible/induction: able to be induced/ being induced
repressible: can be repressed
(B1-4) positive vs. negative regulation-1157
negative: regulated by a repressor
positive: regulated by an activator
positive and negative regulation does not mean increasing and decreasing transcription, however repressor always represses and activator always promotes
eg. CRP+cAMP
(B1-4) transcriptional activator vs. transcriptional
repressor
Activator: bind to DNA and enhance RNAP activity at the promoter
Repressor: bind to DNA to inhibit RNAP binding to the DNA. regulated by effector
eg. allolactose is the effector for the lac repressor protein
(B1-4) specificity factor
makes the RNAP bind to a specific promoter sequence
eg. TBP makes RNAP bind to promoters with a TATA box
sigma 70 makes it bind to promoters with -35/-10
(B1-4) promoter vs. operator
Operator is where repressor binds
promoter is where RNAP binds.
(B1-4) effector (i.e. small molecule cofactor)
regulates the binding of the repressor/activator to DNA
eg. cAMP->CRP
allolactose->lac repressor
(B1-4) allostery (conformational change)
The binding of the effector to the repressor’s allosteric site changes the conformation of the repressor, releasing it from the DNA
(B1-4) diffusible factor (trans-acting) vs. operator (cis-
acting) -1159
diffusible factor: things like repressors or activators that will act on all strands. (trans: other)
Operator: changes to the operator will only affect the gene that the operator is on. (cis=same) it acts on the same strand
(B2-1) glycosides
Sugar linked to a functional group via anomeric bond at the 1-carbon replacing the H of the OH. If it the orientation is above the plane, then it is an alpha-glycoside and if it is below the plane then it is a beta-glycoside.
(B2-1) galactose vs. glucose vs. arabinose
Galactose vs Glc: Galactose has a different orientation of the hydroxyl group at the 4-carbon
Arabinose: the isomer of glucose. Straight chain, and an aldehyde
(B2-1) glucoside vs galactoside
glycoside with a glucose and a galactose respectively
(B2-1) isomerization
INTRA molecular rearrangement of electrons that result in an isomer of the original and the overall oxidation states remain the same
(B2-1) transglycosylation
the mechanism used to form glycoside bonds and is how beta-galactosidase creates allolactose
(B2-1) lactose vs. allolactose
lactose is a sugar created by the dehydration of glucose and galactose.
(B2-1) thiogalactoside
thiogalactoside transacetylase detoxifies the cell by acetylating non-metabolic pyranosides to remove them from the cell.
(B2-1) IPTG
substitute for allolactose in lab setting, it is made of galactose and substituting the OH on the 1-carbon with a sulfur-methyl.
(B2-1) gene vs gene product
Gene is the DNA the codes for the mRNA equivalent of the product
gene product: is either the fRNA or the protein that the DNA codes for
(B2-1) lac operon
combination of three genes that are required to use lactose as a carbon source, its promoter and operator. The Lac I gene is not part of the lac operon because it has its own promoter.
(B2-1) lacZ gene vs beta-galactosidase
Lac Z is the first gene in the sequence, and codes for beta-galactosidase which hydrolyzes lactose to form glucose and galactose. Allolactose is also a minor side product.
(B2-1) lacY gene vs beta-galactoside permease
Lac Y codes for beta-galactoside permease which faciliates intake of lactose
(B2-1) lacA gene vs beta-galactoside transacetylase (thiogalactoside transacetylase)
Lac A, the last gene in the operon. Processes toxic galactose, to remove from the cell.
(B2-1) O1 vs. O2 vs O3 lac operators
O1: between the promoter and the lac z gene and is the tightest bond.
O2: is inside the lac Z gene
O3: inside the lac I gene
(B2-1) lacI gene vs lac repressor
Lac I repressor: a tetramer made of 4 identical monomers, the gene product of Lac I
binds to the operator to inhibit transcription of the lac operon. Conformation changes if allolactose binds to the allosteric site.
(B2-1) cAMP receptor protein (CRP / catabolite activator protein / CAP)-1165
activator for secondary sugar operons.
homodimer with binding sites for DNA and cAMP
(B2-1) inducer
molecules that bind to repressors/activators to regulate gene expression
eg. allolactose is the inducer for the lac operon
(B2-1) cyclic AMP-1165
co-activator for CRP. binds to CRP to create a complex which can bind to DNA to induce transcription
(B2-1) catabolite repression
regulatory mechanism to choose a more favourable source of over other alternative sources.
(B2-1) regulon
network that share a common regulator
eg. cAMP/CRP are common to the secondary sugars- lactose and arabinose.
(B2-2) DNA HELIX STRUCTURE
(B2-2) Sources: NnC 8.2 p288-289 Fig. 8-13
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(B2-2) helical turn
Helical turn: 1 complete physical turn in the DNA.
positive coil will be tighter, thus less nucleotides per turn.
(B2-2) right-handed
Form A and B are right handed (counterclockwise going up) in regards to helical sense.
(B2-2) axis
The center which the DNA rotates/spiral around. The B-form (watson-crick) lines up with the axis perfectly. The A-form is tilted around the helical axis.
(B2-2) base-stacking
base stacking interaction help stablize the DNA and mantain its double helix structure.
(B2-2) planar / aromatic ring
aromatic ring= benzene ring
(B2-2) major vs. minor groove
Major: 4 functional groups on one side- allows for more specificity
minor: 3 functional groups on one side
(B2-2) hydrophilic vs. hydrophobic
hydrophilic: water loving (polar)
hydrophobic: water-hating (non polar)
(B2-4) (structural) motif / fold / supersecondary structure-1162
small recognizable structural characteristic of folding patterns in amino acids.
made of small beta sheets and alpha sheets and is between a tertiary and secondary structure.
(B2-4) domain
Stable configuration with a specific function. may consist of multiple motifs.
Part of a protein with its own specific function.
(B2-4) topological motif
motif on the outside of a protein that serves to carry out a function
(B2-5) DNA-BINDING PROTEINS
(B2-5) Sources: NnC 28.1 p1160-1162, Fig. 28-9 through 28-13, “ModuleB- LacRepressorStructure”
NA looping
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(B2-5) DNA-binding domain
Domain with motifs such as helix turn helix or zinc fingers that can recognize specific DNA patterns and bind to those regions
(B2-5) recognition / specificity
hydrogen bonds can form between the major(or minor) groove of the DNA bases with parts of the R group of amino acids which allows proteins to recognize specific parts of the DNA.
However, A=T (or C=G) does not only bind to only one amino acid.
(B2-5) recognition helix
called the alpha helix, is a part of the helix turn helix that recognizes sequences in the DNA
(B2-5) functional groups in the major & minor groove of DNA
Major: Methyl, H donor, H acceptor and other H (stray hydrogen)
(B2-5) hydrogen-bond acceptor vs donor
hydrogen-bond acceptor will be an atom with strong electro-negativity. This is how most nucleotide pairs are recognized.
(B2-5) thymine methyl group
This non-polar group at 5-carbon allows thymine to be easily distinguished from cytosine.
(B2-5) protein-DNA binding interaction motifs
most common are helix turn helix and zinc finger. And they interact with the DNA via hydrogen bonding.
(B2-5) helix-turn-helix, zinc finger, homeodomain
helix turn helix: used to recognize specific DNA, it is about 20 nucleotides long. There are two alpha-helices about 7-9 nucleotides long and a beta sheet to connect them. looks like a trapezoid shape.
Zinc finger: a zinc ion stabilizes to 4 amino acids which link to approximately 30 nucleotides which form a loop which interacts with DNA
(B2-6) COMBINATORIAL CONTROL
(B2-6) NnC 28.1 p1163-1165, Fig. 28-14, 28-15; 28.3 p1176-1177, Fig. 28-27; “ModuleB- LacRepressorStructure”
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(B2-6) protein-protein interaction motifs
Proteins often require binding to another protein
eg. RNAP to regulatory proteins/subunits
Thus, there are domains for dimer formation which contains motifs that facilitate such binding.
(B2-6) leucine zipper, helix-loop-helix
leucine zipper: is a amphipathic (hydrophobic and hydrophilic) alpha helix with one side hydrophobic. This allows for the dimer to form. zipper can side.
Helix loop helix: Helix loop helix can bind to another HLH to form a dimer.
(B2-6) protein families
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(B2-6) homodimer vs. heterodimer
Homo dimer: identical proteins that make up a dimer
Hetero dimer: two different proteins make a dimer.
(B2-6) combinatorial control
Genes, especially in eukaryotes, are controlled by more than 1 factor. There are activators and repressors, enhancers and silencers that control the expression of a gene.
(B2-7) EUKARYOTIC TRANSCRIPTION ACTIVATORS
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(B2-7) enhancer / upstream activator sequence (UAS)
UAS: Region farther away from the promoter, is another regulatory region
(B2-7) basal transcription factor vs activator vs coactivator (proteins)
Basal (general) transcription factors:
(B2-7) Mediator (complex)
Large protein with 20-30 polypeptides that binds tightly to CTD of RNAP initiation complex and activator.
(B2-7) architectural regulator (protein) and DNA looping
sometimes the activator and the inr sequence are far apart. The architectural regulator will bind to the DNA and fold it to bring the inr closer to the activator which allows the mediator to connect the transcription activators on the UAS with the RNAP on the inr.
(B2-7) Sources: NnC 28.1 p1158, Fig. 28-5; 28.3 p1177-1179, Fig. 28-28, 28-29; “ModuleB- ActivatingElements”
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