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Biology 200

Molecular cell biology

QuestionAnswer
What is the major difference between prokaryotes and eukaryotes? comparmentalization (nucleus and nuclear pores, molecules movement)
What is transcription? process by which the information stored in DNA is copied into RNA for eventual use
What is RNA processing? From pre-mRNA to mRNA by removing sequences and stabilizing RNA molecule
What is translation? process by which information stored in RNA is used to create a protein of specific amino acid sequence
What are the major DNA properties? 1. It contains all the information required to build the cells and tissues of an organism. 2. The information is stored in units called genes.
What monomers are polymerased in DNA replication? dNTPs (deoxyribonucleoside triphosphate)
What monomers are polymerased in DNA transcription? rNTPs (ribonucleoside triphosphate)
What is a gene? a discrete unit that encodes information that cells require.
What are nucleic acids? They are DNA and RNA, linear polymers of monomers called nucleotides
What is the backbone of nucleic acids? It is a pentose phosphate backbone (deoxyribose in DNA and ribose in RNA)
What are the purine bases? Adenine and Guanine (Ah God c'est pur!)
What are the pyrimidine bases? Cytosine, thymine and uracil (c'tu pyrimidine?)
Which pyrimidines are in DNA only? in RNA? DNA : cytosine and thymine RNA : uracil
How each monomer of the backbone are linked together in nucleic acids? By phosphodiester bonds
How could we qualify the transcription by RNA polymerases? it is SIMULTANEOUS.
Where are exactly the phosphordiester bonds? They link the 3' hydroxyl and the 5' phosphate of two sugars from two nucleotides.
Where are linked the bases on a nucleotide? They are linked on the first carbon of each
What is the length of most RNAs? Most RNAs are <100 to 10 000 nucleotides long
What is the length of most cellular DNA molecules? They can be 100 000 000 nucleotides long.
Is the linear pentose phosphate backbone charged? Yes, it is negatively charged.
What is the interaction between bases in a base pair? Hydrogen bonds.
Are there free groups on a nucleic acid molecule? Yes. Free 5' phosphate group and Free 3' hydroxyl group.
Is there a directionality in nucleic acids reading? Yes. Always from 5' to 3'
What is the stability difference between DNA and RNA? DNA's deoxyribose (pentose) has a H on the 2nd carbon : stable. RNA's ribose (pentose) has a OH un the 2nd carbon : unstable (because it can engage in nucleophilic attack on the phosphodiester bond)
On what form do we find nucleic acids in vitro? DNA : double-stranded, helical. RNA : single-stranded
In natural DNA, how base pairing works? It follows the Watson-Crick base pairing : A always bonds with T and G always bond with C
Is it possible for a DNA molecule NOT to respect the Watson-Crick base pairing? Yes. In laboratory, we can produce G-T and C-T base pairs.
Can G-U base pairs exist? Yes, in double-helical of otherwise single-stranded RNA.
On which structure do we find DNA in the cell? B-DNA
What is the difference between the two alternative structures of DNA? A-DNA is a more compact form (more bases per turn). It doesn't respect Watson-Crick base pairing. It can be whether RNA-DNA of RNA-RNA
Interactions between two polynucleotides forming a double helix are parallel or antiparallel? Antiparallel
How many H bonds are there between T and A? Between C and G? 2 H bonds between T and A, 3 h bonds between C and G.
Why is DNA double helix flexible? Because there are no hydrogen bonds parallel to the axis of the DNA helix.
Can DNA be bent? Yes, by DNA binding proteins.
DNA bending is necessary for it to...? ...be packed in chromatin!
What is the TATA-box binding protein? It is a protein that binds to promoters of most eukaryote genes. This step is necessary in the transcription process.
Why denaturation has to occur? is it a reversible process or not? It is necessary for replication and transcription because it gives access to information. Yes, it is reversible.
How can DNA denaturation occur? By the disruption of hydrogen bonds between bases or by repulsion of negative charges on phosphate of DNA strands.
What is the relation between T and absorption of 250-nm light in single-stranded DNA? at lower temperature, ssDNA can react with itself and absorb less light. As we increase T, intermolecular interaction, including with ssDNA itself, are broken and light absorption increases too.
What is the relation between T and absorption of 250-nm light in double-stranded DNA? dsDNA absorbs less light at low T because bases are stacked. When we increase T, we denature DNA, which becomes single-stranded and absorbs more light.
What is the Tm? Temperature of melting is when 50% of the base pairs in double-stranded DNA are denatured.
Is there a relation between Tm and the number of GC pairs in a genome? Yes. GC base pairs are more stable (3 H bonds) and need more T to denature. Consequently, Tm is higher when there are more GC pairs within a genome.
What are the factors that can lead to DNA denaturation? Raising temperature, Reduce ionic concentration, extremes of PH, adding agents that destabilize hydrogen bonds ( formamide or urea)
What happens with single-stranded DNA when denaturation conditions are reversed? They stick back together to renature into double-stranded DNA molecules. (ATTENTION : single-stranded DNA molecules have to be in contact with complementary strands)
Where do we find circular DNA molecules? In prokaryotes and viruses
What occurs in circular DNA when binding proteins bend a certain section? It causes a torsional stress : supercoiled
How can a cell react to supercoiled? Topoisomerase l can create a nick on one strand which will bring back the relaxed DNA circle. Topoisomerase ll causes nicks on both strands and can ligate 2 circular DNA molecules.
What is a hairpin loop? It occurs in ssRNA, between complementary sequences, antiparallel base pairing of 10-14 nt.
What is a stem loop? antiparallel base pairing of more than 10-14 nt, 3D double helical stem region.
What is a pseudoknot? it is one of the tertiary structures of RNA : it contains 2 stem loops.
How do we call RNA with catalytic properties? A ribozyme. some ribozyme can catalyze self-splicing!
RNA polymerase reads the template strand from which end to which end? from 3' to 5'
RNA is synthesized from which end to which end From 5' to 3'
Is pre-mRNA identical to the template strand? No. It is identical to the non-template strand (U instead of T)
RNA polymerase joins incoming rNTPs to previous one by creating phosphodiester bonds. Why are these bonds favourable? Because they have a lower energy than triphosphate bonds between alpha Phosphate and beta Phosphate.
Where is the transcription initiation start site? At +1. Nothing up of +1 is transcribed.
What is upstream the transcription initiation site? Non-coding sequences ( e.g. promoters to regulate gene expression and to recruit RNA polymerase for transcription initiation ) that recruit activators or repressors and other regulators.
What is downstream the transcription initiation site? coding sequence for a primary RNA transcript.
Are many stages of transcription are there? 3 : initiation, elongation, termination.
Describe briefly the initiation stage. RNA polymerase binds to promoter sequence, locally denature DNA and catalyzes the first phosphodiester linkage.
Describe briefly the elongation stage. polymerase advances from 3' to 5' down the template strand, denaturing DNA and polymerizing RNA.
Describe briefly the termination stage. RNA polymerase recognize a stop site, releases the complete RNA and dissociates from DNA.
When occurs the ''closed complex''? When RNA polymerase binds to promoter sequence in duplex DNA
When occurs the ''open complex''? When RNA polymerase melts duplex DNA near transcription start site.
What is the transcription bubble? It is what open complex looks like when RNA polymerase melts duplex DNA.
What are GTFs? When and where are they useful? General transcription factors. They are proteins that help eukaryotic RNA polymerase to find promoters and initiate transcription.
Is melting of duplex DNA a ATP-dependent process? Yes.
If RNA polymerase leaves the promoter region, in which transcription stage are we? Elongation
When is nascent RNA exiting polymerase? In elongation stage of transcription.
Why RNA polymerase doesn't fall off the template strand during elongation? Because DNA-RNA hybrid region is very stable.
What is the length of the longest gene? How long does it take to transcribe? millions of nt. 24h of transcription.
What is the speed of elongation? 1000 nt/min
Can many polymerases transcribe the same gene at the same time? Yes. As soon as the promoter sequence is free, another RNA polymerase can join and initiate transcription.
In eukaryotes, is the primary transcript functional? No. it has to be processed.
How many subunits are there in E. Coli RNA polymerase? 5.
Do all polymerases resemble E. Coli RNA polymerase? Yes.
Polymerases are highly conserved. How? Why? It means that their identity in nucleotide sequences is conserved. Because they deal with conserved DNA and RNA and catalyze the same reactions.
What happens with DNA when entering the enzyme (polymerase) It sharply bends.
How are genes organized in prokaryotes? Genes with a common function are often arranged linearly in operons and transcribed together on a single mRNA. DNA is circular
Are there non-coding gaps in DNA of prokaryotic genomes? Only a very few.
Is there a nuclear membrane in prokaryotes? What consequences this has on the speed of response to environment changes? No. This allows the organism to respond very rapidly to environmental changes/fluctuations
What is special about transcription and translation in prokaryotes? It can occur at the same time since prokaryotic RNA doesn't need to be processed.
Does the prokaryotic RNA needs to be processed No, because prokaryotic DNA only contains a very few non-coding sequences.
In E. Coli, what are E, D, C, B and A? They are enzymes/proteins required for the stepwise biosynthesis of tryptophan
What happens in E. coli cell when there is less trp? It activates transcription of all genes (EDCBA) required for biosynthesis of trp.
How do we call non-coding gaps in DNA? Introns
Is it possible for a mRNA to contain more than one start site? Yes, in prokaryotes, mRNA contains information for the synthesis of more than one protein at the same time : it contains a start site for each protein to be produced.
How is regulation in prokaryotes It is called co-regulation : genes that codes for related proteins are driven to transcription by a single promoter that leads to transcription of all enzymes needed for biosynthesis.
Ho are genes organized in eukaryotes? Genes that encodes for proteins that work commonly are scattered on several chromosomes. (BUT some can be on the same chromosome)
Is there co-regulation in eukaryotic DNA? No. Transcription initiation with different promoters : regulation is not achieved simply by physical linkage.
What is the first step of RNA processing? The 5' cap structure (7-methyl-G) is added to the 5' end of nascent RNA.
How is the cap structure linked to the 5' end of nascent RNA? It is linked to the terminal nucleotide by a 5'-5' linkage.
What is the polyadenylation? In RNA processing, it is the step when 100-250 A residues are added to the 3' end of pre-mRNA.
What is the major step in RNA processing? Removal of introns and ligation of exons.
Can the RNA transcript be sliced differently? Yes. It leads to the synthesis of different forms of the proteins encoded by the gene.
Name an advantage of alternative splicing of the primary transcript. It increases protein diversity.
Are all the transcription factors that regulate expression of a gene close to the promoter region? No. They can be tens of kb upstream or downstream the promoter region.
What is the use of the 5' cap structure? It provides protection and stability to the primary transcript.
What is the use of the poly-A tail and what proteins adds this tail to the primary transcript? It provides stability to the primary transcript and it is added by the poly-A polymerase.
When do we obtain the mature mRNA? When introns are removed and when exons are linked together.
Give an example of alternative splicing of pre-mRNA Fibronectin gene : a) fibroblast retain ElllB and ElllA to anchor fibronectin to the membrane b) hepatocytes remove ElllB and ElllA to make fibronectin more soluble. So, axons are included or excluded depending of the cell type.
Should exons that are cut off the pre-mRNA be called introns? No, they are still exons, but in alternative splicing.
What is the ultimate goal of regulation of transcription? recruit RNA polymerase ll to the promoter to position its active site over the transcription start site and then, initiate transcription.
What is the goal of protein chromatography? Study a pure sample of a specific protein
How can we isolate a specific protein by chromatography? by doing different kinds of chromatography based on charges, size, linkage to antibodies, etc.
Briefly explain the experience of chromatography with RNA polymerases and sensitivity to alpha-amanitin Good luck!
Do the three eukaryotic RNA polymerases play the same roles? No, they catalyze transcription of genes encoding different classes of RNA.
Give the role of each eukaryotic RNA polymerase l : (in the nucleolus) transcribe precursor rRNA. ll : transcribes mRNAs and 4 small nuclear RNAs (snRNAs for RNA splicing) lll : transcribes tRNAs, 5S rRNAs and other small stable RNAs (including one involved in RNA splicing)
How many subunits are there in bacterial RNA polymerase? 5
How many subunits are there in yeast RNA polymerase ll 12
What is the utility of the w subunit and is it present in both prokaryotic and eukaryotic polymerases? It occurs stability to the prokaryotic RNA polymerase and is not present in eukaryotes.
Are there similarities between prokaryotic and eukaryotic polymerases? Yes. There structure is very similar. The core-complex structure is conserved between eukaryotes and prokaryotes. B and B' subunits are similar (but non identical), alpha-like subunits (related in terms of amino acids) and they share common subunits.
Is there the same level of complexity between eukaryotes and prokaryotes? No. In eukaryotes there's a new level of complexity : additional enzyme subunits for specific roles/functions (transcription and its regulation by factors)
Talk about the subunits in the three eukaryotic RNA polymerases They all have 2 large subunits and 10-14 smaller subunits each (some are common to 2 or all 3 polymerases)
Do all subunits need to come together to form a functional RNA polymerase? Yes.
What is important about the largest subunit (Beta) in RNAP ll? It has a carboxyl-terminal domain (CTD)
What is the structure of the CTD in mammals? It is made up of 52 nearly identical repeats of Tyr-Ser-Pro-Thr-Ser-Pro-Ser
What is the difference in CTD of RNA polymerase molecules that initiate transcription (1) and those that actively transcribe (2)? (1) are unphosphorylated and (2) are phosphorylated
What is the pre-initiation complex necessary for? Transcription initiation
What contains the pre-initiation complex? RNA polymerase and many general transcription factors (TFll for factors associated with RNA polymerase ll)
In what environment can transcription initiation be studied? In vitro AND in vivo
What are the enzymes responsible of desphosphorylation of the CTD of RNA polymerase? Kinases and phosphatases
What is the most common and efficient promoter on the eukaryotic DNA? The TATA box
What is the TATA box ? A promoter sequence on eukaryotic DNA that is made of As and Ts (requires less energy to melt) like most promoters
What are the core factors? GTFs
What are the TFlls and what do they do? They are the Transcription factors for RNA pol ll. They associate with RNA pol ll at the +1 and in the promoter region.
What is the first step of the formation of pre-initiation complex? recruitment of TBP. this protein binds to TATA-box in AT-rich region of a minor groove and bends DNA. In vivo, this step requires TFllA, which forms a complex with TFllD and the TATA-box.
What is the second step of the formation of pre-initiation complex? Recruitment of TFllB. this factor binds to DNA and to TBP (interaction with both)
What is the third step of the formation of pre-initiation complex? Recruitment of TFllF. This factor binds to preformed complex of RNA pol ll. it is a tetramer (4 subunits) and this new complex then binds to DNA. Note : RNA pol ll does not bind precisely at the TATA-box.
What is TFllD? In vivo, it is a complex of TBP and 13 other subunits called TAFs (TATA-binding associated factors)
What is the forth step of the formation of pre-initiation complex? TFllE (a tetramer) binds next. it creates a docking site for TFllH
What is the fifth and last step of the formation of the pre-initiation complex? Recruitment of the TFllH. This factor is a helicase that locally unwind DNA and allows the Pol ll to form the open complex.
After the building of the pre=initiation complex, what allows the phases of transcription and elongation to begin? TFllH's kinase (subunit) phosphorylates CTD tail of RNA pol ll which promotes elongation.
What are DNA microarrays? It is a method to study the genome. By putting sequences of DNA on a glass slide or ''gene chip'' and then by putting these samples into different environment, we can study, thanks to different dyes, if gene sequences are expressed or not.
In prokaryotes, what is the transcriptional control? It if the fact to regulate gene expression in response to changes in environment. Repression : little or no mRNA synthesis. Activation : up to 1000x or more mRNA are synthesized.
Describe transcriptional regulation in unicellular organisms 1. Acutely sensitive to changes in temperature (physical) or nutrient levels (nutritional), etc. 2. produce immediately the protein/enzyme and factors required for the survival of the cell in a given environment
Describe transcriptional regulation in multicellular organisms 1. Certain genes gave to be turned down at certain times in certain tissues for development of other organs, structures, tissues. 2. coordination of processes for normal development rather than rapid response.
What is an operon? In prokaryotes and bacteria, It is a sequence of genes that encodes for proteins that work together
In prokaryotes and bacteria, how can we turn/off a gene? It depends on interplay of interplay between repressors and activators of RNA polymerase
What is the utility of E. coli lac operon It encodes 3 enzymes that are required for catabolism of lactose (creates energy for the bacteria)
Are all the genes of E. coli organized into operons? No, about half of them are organized into operons.
What is the use of trp operon? It encodes 5 enzymes required for the biosynthesis of tryptophan.
To initiate transcription, E. coli RNA polymerase must associate with what? a sigma factor
In E. coli, where the sigma 70 factor binds? In the promoter region
In E. coli, where the lac repressor binds? in the operator region an blocks start site.
What happens to transcription of lac operon in E. coli when lactose is absent? Repressors cannot bind to lactose, because there is no lactose, so they bind to the activator region and block the start site.
In E. coli, what happens to transcription of lac operon when lactose is present? Lactose binds to lac repressor and removes it from operator sequence.
What is the de-repression of transcription of the lac operon? It is when lactose binds to the repressors, removing them from the operator sequence and allowing transcription.
In prokaryotes and bacteria, how can we turn on/off a gene? it depends on interplay between repressors and activators of RNA polymerase.
What is the utility of E. coli lac operon? It encodes 3 enzymes that are required for catabolism of lactose (a source of energy for the cell)
Are all genes of E. coli organized into operons? No. about half of them are organized into operons.
What is the use of trp operon? It encodes 5 enzymes required for biosynthesis of tryptophan.
To initiate transcription, E. coli RNA polymerase must associate with what? a sigma factor
In E. coli, where the sigma factor binds? In the promoter region.
In E. coli, where the lac repressor binds? In the operator region an blocks start site.
What happens to transcription of lac operon in E. coli when lactose is absent? Repressors cannot bind to lactose, because there is no lactose, and they bind to the activator region and block start site.
What happens to transcription of lac operon in E. coli when lactose is present? Lactose binds to repressors, remove them from the activator sequence, which allows transcription of lac operon
What is the de-repression of transcription of E. coli lac operon? It is when repressors are removed from the activator sequence.
Are de-repression and activation of E. coli lac operon transcription the same thing? No.
What is the best source of energy between glucose and lactose (or other complex sugars)? glucose
What happens with operons like E. coli's lac operon when glucose levels are low? transcription of lac operons (or others) is activated.
What is synthesized by E. Coli when glucose levels are low? cyclic AMP
Where the cAMP binds when glucose levels are low in E. coli? It binds to a transcriptional activator protein called CAP.
In E. coli, where does the CAP bind when glucose levels are low? What is the condition for CAP to actually bind to this site? It binds to the CAP site. It has to complexed with cAMP.
In E. coli, What is the effect of CAP-cAMP complex It interacts with RNAP and greatly stimulates the rate of transcription initiation.
What does CAP mean? catabolic activator protein.
What is the action of sigma factors? They recognize specific DNA sequences as promoters and recruit RNA polymerase.
What happens with sigma factors once transcription is initiated? They are released.
Which sigma factor is the best known? sigma 70
What sequences the sigma 70 factor recognizes? TTGACA ....... TATAAT
Which promoters are recognized by sigma 54? promoters of genes encoding enzymes required for nitrogen metabolism.
In prokaryotes, is the operator sequence transcribed? Yes, and it is part of the untranslated region of the transcribed sequence.
What is the most important characteristic of the TATA box? It is an AT-rich region.
What is the equivalent of the TATA box in prokaryotes? the TATAAT box.
Where starts the TATA box for genes that are transcribed at high levels? 35 bp upstream the start site.
What is the major role of the TATA box? positioning RNA polymerase for transcription initiation.
In eukaryotes, what happens when we delete sequences between +1 and 25-35 bp upstream without affecting TATA box? Why? Reduction of transcription initiation or complete deletion of it. RNA pol occupy a certain area and needs space to bind to the TATA box.
Are there alternatives to the TATA box? Yes. 1) Some genes have an initiator element which includes C at -1 position and A at +1 position. 2) Some gene contain a CG-rich stretch of 20-50 bp (called CpG island) within about 100 bp of the start site region.
Is the TATA box a promoter? Yes, but some alternative sequences can also play this role.
What are the promoter-proximal elements? They are sequences near the promoter region (within 100-200 bp of the promoter) that can regulate transcription.
Can the promoter-proximal elements be cell-type specific? yes.
Name a good way to find the promoter-proximal elements Linker scanning mutations :
True or false : all genes of the mammalian genome use TATA box or initiator elements? False : 60-70 of genes in the mammalian genome do not use TATA box or initiator elements.
Are the promoter-proxiamal elements par of the enhancers? Are they always close to the promoter? Yes. No, they can be far from it.
Is the bacteria's CAP a promoter-proximal element? No, but it plays the same role.
In liner-scanning mutation (technique to find the promoter-proximal elements) wat is the location of the control region (containing promoter-promixal elements)? 100-150 bp upstream a start site
Resume the linker scanning mutation technique that is used to find promoter-proximal elements in the control region. We remove some sequences of nt in the control region and thanks to the reporter, we can see if there is or not a diminution of mRNA production.
In eukaryotes, can elements that regulate transcription of a gene span kb? Yes. Transcription factors that regulate gene expression can bind to sites that are tens of kb upstream or down stream of the promoter.
What is the deletion analysis useful for? identifying and mapping enhancers.
Where can be located enhancers? The can be >50kb from the gene that they regulate.
Are enhancer cell-type specific? Yes
What is the fist step of deletion analysis in order to map and identify enhancers? We delete sections from the 5' end to a certain point upstream the start site. We then obtain different ''5' deletion series''.
In deletion analysis (to find enhancers) what do we do after obtaining different 5' deletion series? We ligate eache series to a plasmid vector (E. coli) containing a reporter gene. E. coli is transformed and we isolate each kind of plasmid DNA obtained (5'-deletion mutants)
In deletion analysis (to find enhancers) what do we do with the different 5'-deletion mutants that we have created? We transfer each type of 5'deletion mutants separately into cultured cells.
In deletion analysis technique (to find enhancers) what contains the cultured cells once we have added the 5'-deletion mutant? it contains reporter plasmid, the reporter mRNA and the reporter enzyme (in different quantities depending of the importance of the sequence that has been deleted)
What is the final step of 5' deletion technique (to find enhancers) We prepare cell extract and we assay activity of reporter enzyme.
Resume the experience with B-globin and plasmids that proves the importance of enhancers 1. create to plasmids (one only containing the b-globin gene and another containing b-globin gene and SV40). We manipulate mRNA produced by both plasmids and, by chromatography, we see thatmuch more b-globin mRNAs produced by the cell containing SV40
Is the distinction between enhancers and promoter-proximal elements clear? no.
Name transcriptional control elements in eukaryotes TATA box, enhancers, promoter-proximal elements, exons and introns
What is UAS? Upstream activating sequence. This sequence is found in many yeast genes and works like an enhancer.
In yeast, where is the TATA box? about 90 bp upstream from the start site.
Name the promoter elements TATA box, initiator elements, CPG island
Name the enhancer elements promoter proximal and enhancers
Name to ways to identify control sequences on DNA linker scanning or deletion analysis.
Activators and repressors of transcription are protein or nt sequences? They are proteins
Name to ways to identify activators and repressors 1) DNase l footprint 2) Electrophoretic shift assay (also called EMSA or ''gel shift'')
Briefly explain how proteins can be separated by chromatography A solvent is continuously applied at the top of the column after applying the sample. With time, proteins go down the column on a solid matrix fall off the column into a tube by porous plug.
Name the 3 major types of matrix for chromatography a) ion exchange chromatography b) gel-filtration chromatography c)affinity chromatography.
What is the utility of DNase l footprinting experiments? 1) reval specific binding sites for DNA binding proteins and 2) can be used as an assay in transcription factor purification.
Explain briefly how DNase l footprinting experiment works 1. add DNase l to samples of DNA containing or not the DNA-binding protein. 2. separate all fragments of samples by electrophorese. In samples containing the protein, there is a blank in the electrophorese fraction. 3.identify the protein-binding sequence
What is the advantage of using EMSA instead of footprinting? it is a better technique for quantitative analysis of DNA-binding proteins.
What is the disadvantage of EMSA technique? It doesn't provid the specific DNA-binding sequence.
Explain briefly how EMSA technique works We use the same segment of DNA for each fraction of the electrophorese. A segment of DNA bound to a protein will migrate slower in a gel than the DNA alone. We can
What is the difference between promoter elements and enhancer elements? Promoter elements help recruit RNA polymerase and enhancer elements help and regulate the whole process.
What are the major steps of purification of transcription factors? 1) map binding site (by footprinting exp.) 2) synthesize multiple copies of DNA sequence, couple it to beads 3) incubate nuclear extract with beads, wash, elute (how?) 4) Use EMSA 5) test if the protein stimulates transcription
Give an example of in vitro assay for transcription activity By adding SP1 can stimulate transcription from the SV40 promoter but has no effect on another viral promoter.
In the in vivo assay for testing the efficiency of a transcription factor, what corresponds to protein X? The transcription factor that we have purified.
Explain the co-transfection assay (in vivo) We add in the nucleus of a cell two plasmids. 1 : contains the sequences encoding the transcription factor 2 : contains a reporter gene and a binding site the transcription factor. transcription factor binds to plasmid 2 and reporter gene is expressed.
In co-transfection assay, why is it important that the cell lacks or does not encode the gene encoding the protein to be tested? Because if the cell already produces the transcription activator on its own, you will not know if the sequence that you have isolated ACTUALLY encodes for this transcription activator.
What is a transcriptional activator? It is a modular protein that have distinct functional domains : 1) DNA binding domain 2) activation domain, which interacts with other proteins to stimulate transcription.
Can the co-transfection assay work for testing a repressor? Yes.
What is the first experiment that has been driven to prove the modular characteristics of an activator? The co-transfection experiment with GAL activator and lacZ gene in yeast. But it is not just the classic co-transfection experiment : by removing (in vitro) certain sequences of the gene encoding the activator, we can prove that it really contains a bindi
Are transcriptional repressors the most common way to regulate transcription of genes? No. transcriptional activators are much more used than transcriptional repressors.
What are transcriptional repressors In eukaryotes, they are the functional converse of transcriptional activators.
Are transcriptional repressors modular proteins? Yes. instead of an activation domain (activators) they have a repression domain and they also have a binding domain.
What is the effect of chromatin remodelling complexes when they interact with transcriptional repressors? They pack the DNA up, which doesn't get access to TFllD complex to the promoter sequence and there is no transcription.
Can the binding domain of a transcriptional activator of repressor bind to any DNA sequence? No. They contain specific structure motifs that bind to specific DNA sequence.
How are called the DNA sequences to which DNA-binding domains of repressors or activators bind? Enhancers or repression sequences.
Explain the interactions that explain specificity of binding domains of activators or repressors? It results from non-covalent interactions between alpha helices of DNA-binding domains and atoms on bases in the major groove of DNA.
What is the common characteristic of zinc-fingers motifs? the primary structure of these DNA-binding motifs folds around the zinc ions in a finger-like shape that can then interact in a sequence-specific way with DNA.
name two types of zinc-finger motifs? C2H2 zinc finger and C4 zinc finger.
True or false? The C2H2 zinc-finger motifs are the most common binding motifs in plants? False. in humans.
In the C2H2 zinc-finger motifs, what are the 4 residues that binds with the Zn ion? 2 cysteine and 2 histidine.
In the C2H2 zinc finger motifs, what allow the DNA-binding domain to actually interact with DNA? The cysteine and histidine residues compact the DNA-binding motif into helices, and this compact form of DNA-binding motif can than be inserted into the major groove of DNA.
Name the major difference between C2H2 zinc-finger motifs and C4 zinc finger motif C2H2 binds as monomers and C4 binds has homodimers.
In C2H2 zinc-finger motifs, do all alpha helices interact with the DNA? No. in the image, Finger 1 doesn't interact with the DNA.
Name three types of DNA-binding domains zinc-fingers, leucine-zippers and basic helix-loop-helix
Why leucine-zipper called this way? they have a leucine residue at every seventh position.
Name the 3 major characteristics of leucine-zipper proteins 1) leucine residue at every 7th position 2) bind DNA as dimers, ofter heterodimers 3) related proteins have different hydrophobic amino acids.
What is the term for the larger family of proteins like leucine-zipper? basic-zipper or bZip.
What distincs basic helix-loo-helix from leucine-zipper ? a nonhelical region between two alpha-helical regions.
can we really have ONE transcriptional factor for each gene of the genome? NO.
Name three elements that allow transcription factors a regulatory specificity. 1) heterodimers (3 monomers can forme 6 dimers) 2) inhibitory factors 3) cooperative binding.
Give an example of cooperative binding NFAT and AP1 hardly bind to their DNA-binding site separately but, when they act together, they bind very hardly to their DNA-binding site.
In interactive binding, name the two interactions that occur. Binding proteins interact with their DNA binding domain AND with each other.
Where is oestrogen receptor located? On the plasma membrane
How work the binding domain of the oestrogen receptor? When eostrogen is present, it binds to the receptor, which makes a alpha helix bind to the ligand. The receptor then adopt a new conformation which is hydrophobic : the amphipathic subunit of the cofactor can bind to the complex.
What is an enhanceosome? Give an example. It is a large DNA-binding complex that assemble from transcription factors as they bind to their multiple binding sites in an enhancer. Example : B-interferon enhancer.
What is the mediator complex? It is a huge series of proteins that bind together as a complex, which always has head, a middle and a tail. Stimulates assembly of pre-initiation complex. It (head and middle) interacts with RNA pol ll and with activation domains of activators.
What is the major role of the mediator complex? It mediates the interactions between enhanceosomes and RNA pol ll
What is the action of the post-transcriptional mechanisms genes? they protect vulnerable nascent transcript from degradation and they also process it (to lead the transcript to get protein-coding regions).
What is the action of the endonuclease in RNA processing? It cleaves the transcript at the poly(A) site, removing UTR and adding a poly(A) tail.
Is there UTR at both 5' end and 3' end? Yes.
What is the result of RNA pol ll CTD phosphorylation? The phosphorylated CTD recruits factors required for RNA processing (such as Caping enzymes) and RNA stabilization.
What is the first end of mRNA that goes out from RNA pol ll? 5' end
Can RNA be alone? NO. IT IS NEVER ALONE. It is always associated to different groups of proteins from transcription to translation.
What is the first step in eukaryotic mRNA processing? Addition of the 5' cap end.
How long the nascent transcript has to be for allowing the 7-methyguanosine to be added to the 5' end? 25 - 30 nt long.
What are hnRNPs? proteins that associate with pre-mRNA. Heterogeneous ribonucleoprotein particles
What is the RRM? Describe it. It is the RNA recognition motif, the most common RNA binding domain. It contains 80 amino acids and RNP1 and RNP2, which are motifs that contact the phosphate of RNA. It also contains beta sheets and alpha helixes.
Apart from the RRM, name other RNA-binding motifs RGG box and the KF motif
True or false : RNA-binding motifs interact with backbone of RNA? Name the other major characteristic of RNA-binding domains True. They fold onto themselves to form hydrophobic or hydrophilic pockets that can interact with RNA.
Which molecules keep pre-mRNA from folding on itself? hnRNPs.
In the nuclei, are pre-mRNA processed at the same place where they have been produced? No. They have to be brought to certain centres within the nuclei : DNA and mRNA are separated and occupy specific zones within the nucleus.
When pre-mRNA is produced, is it processed separately from other pre-mRNAs? No, pre-mRNAs are concentrated in different sites and processed at the same time.
How could you describe the CTD of RNA pol ll? It is unfolded and very long.
Why, in many eukaryotes organisms, the CTD tail of RNA pol ll has evolved to become very long? Because while the pre-mRNA is being produced, transcription is so long (with long genes) that it has to be processed at the same time. So, CTD tail also associates with splicing and polyadenylation factors while RNA pol ll is transcribing.
How EM imaging provided evidence for RNA splicing? By putting a processed RNA with the DNA containing the sequence coding for this RNA, we see that both molecules hybridize but the DNA forms certain loops, which correspond to the regions that have been removed from the sequence (introns)
In RNA, which two bases do we almost always find at the 5' end of an intron? And at the 3' end? 1) GU 2) GA
Name the three elements of an intro that never change and that are highly conserved Th GU at the 5' end, the AG at the 3' end and the branch point (A), which is upstream the 3' end. (3' and 5' refer to the intron)
What are the two reactions that characterize splicing of RNA? The two transesterifications.
What happens with the excised lariat intron (after second transesterification) It is degraded.
What are snRNAs? They are part of snRNPs. They are Small nuclear RNAs important for base pairing with the pre-mRNA. (they hybridize with it)
Where do U1 snRNA interact with pre-mRNA ? At the 5' end of the intron.
Why is the branch point excluded while RNA processing? Because it facilitate the first transesterification reaction.
Can RNA-RNA interactions do splicing? Yes.
In self-splicing group ll introns, is there any protein required for splicing? No, it happens only thanks to the RNA catalytic properties.
How many different snRNAs interact with pre-mRNA, with one another and with proteins to form the spliceosome? 5. (U1, U2, U4, U5, U6)
What are endonucleases? Enzymes that cut nucleic acids into shorter fragments.
Describe the action of the spliceosome. It catalyses 2 transesterification reactions that join the exons and remove the intervening intro as a lariat structure, which is degraded.
what are the only mRNAs that are not polyadenylated? the histone mRNAs.
In polyadenylation process, what are the sequences on mRNA that are critical bounding sites? From 5' to 3' : 1) the poly(A) signal rich in A and U repeats 2) the poly(A) site 3) the poly(A) signal rich in G/U.
When 3' end of RNA is processed (cleavage and poly(A)), is it linear? No, thanks to proteins that bind to specific sites and interact together
What is the bigger complex : the pre-initiation complex or the complex for poly(A) of mRNA? They are the same size.
What is the most important step in polyadenylation of mRNA? When PAP (poly(A) polymerase) joins the complex.
What is the major role of PAP? Extend the poly(A) tail at the 3' end of mRNA.
What is the major role of PABPll? It binds to poly(A) of mRNA and it stimulates PAP to work at a higher pace. It also tells it when to stop.
What do SR proteins do? They bind to enhancer sites of the exons and this helps define true splicing sites. They tell the splicing enzymes where to go
Give an example of regulation of mRNA splicing Sex determination in drosophila.
group ll self splicing introns have a structure similar to... spliceosome's snRNA's
How is called the process by which sequence-specific RNA binding proteins can promote or block recognition of a splicing site by spliceosome? alternative splicing
what means mRNPs? mRNA ribonucleotide particles
how many layers does contain the nuclear envelope? To what is linked each part? two : outer linked to endoplasmic reticulum and inner linked to chromosomes.
what complex is located between the two layers of nuclear envelope? nuclear pore complex (NPC)
what is the size of NPC? 125 megadaltons or 30x bigger than a ribosome.
NPC is composed of how many proteins in yeast? in vertebrates? about 50. about 100
What is a maximal size for a molecule to diffuse freely through the NPC? about 60kDa
Can big molecules and multimolecular complexes diffuse freely through the NPC? No, they have to be actively transported.
What do NPC look like? Baskets
Identify on a drawing each part of the NPC see an image. :)
What kind of proteins are transported inside the nucleus? And outside the nucleus? 1) any protein 2) transcription factors, DNA polymerase, enzymes, etc.
Transport through NPC depend on _________ hydrophobic reactions REVERSIBLE (comprendre crisse)
What support the interactions of FG-repeats Y-complexes
What is the role of the matrix of FG-repeats in the center of NPC? blocking the passage of molecules of a certain size.
All nuclear proteins are synthesized in the ________ and imported through ___________ 1) cytoplasm 2) NPCs
Nuclear proteins contain a NLS (what is it?). Is that a DNA sequence or a protein sequence? Nuclear localization signal. Both of them.
What is the characteristic of nuclear proteins that are transported through the nuclear envelope? When they are transported through ER, mitochondria or chloroplasts, do they have the same characteristic? They are transported in a folded state. No.
If we want to add a NLS to a cytoplasmic molecule, would we put it on the 5' end or a the 3' end of the encoding gene? 5' end.
In mRNA, which end has adapted to the regulate the half-life of the mRNA and which end has adapted to the regulation of localization of mRNA (that will lead the the production of the NLS on the corresponding protein)? 1) 3' end 2)5' end.
What happens when we add a NLS to a cytoplasmic target protein? It sends a signal that is very evident to direct other things to the nucleus.
Is there only one NLS that exist? No, there are many NLS.
What is Ran? It is a monomeric G protein.
How many conformations can take the Ran? 2 : one bound to GTP and one bound to GDP
What is the GAP? It is the protein that activates GTPase.
What is linked to GTPase when it is not activated? and when it is activated? 1) GTP 2) GDP
What is the protein required to replace GDP by GTP on the GTPase? GEF ( Guanidine nucleotide exchange factor ) , an activator protein.
In the mechanism of nuclear import, what is the cargo? It represents anything we want to get into the nucleus. (splicing factors, RNA polymerases, etc.)
What a protein must absolutely contain to be imported into the nucleus? A NLS (nuclear, localization signal)
In nuclear import, What is the importin? A protein that contains a receptor that binds to the NLS of the cargo during nuclear import.
In nuclear import, Importin can change conformation whether if it is bound to ____________ or to _____________ 1) cargo 2) Ran GTPase.
In nuclear import, What is the cargo complex? Is it bimolecular or trimolecular? It is a complex containing the Cargo and the Importin. It is bi molecular
In nuclear import, How can the importin can go through the nuclear pore complex? By making transient interactions with the FG-repeats.
in nuclear import, With what does the importin has to interact to let go the cargo into the nucleoplasm? With the GTP-bound form of the ran GTPase.
In nuclear import, What is the main characteristic of the importin once it is linked to the GTPase? It's affinity for the NLS is very weakened dramatically.
In nuclear export, what is the NES? Nuclear export signal.
What do Suttle (navette) proteins have to contain in order to be exported AND imported? NLS AND NES.
In nuclear export, what forms the cargo complex? is it bi or trimolecular? Exportin + GTP-bound Ran GTPaase + Cargo. It is trimolecular.
In nuclear export, what is the main characteristic of the expotin once it is linked to the GTP-bound Ran GTPase? It has now a high affinity to the NES of the Cargo.
Most MRNA export is Ran-independant or dependant? Independant. just a few have to associate with Ran GTPase in order to be exported into the cytoplasm.
What is the name of the exportin that associates with tRNA in order to export it out off the nucleoplasm? Exportin t
Is there a NLS on tRNA? No.
Export of ribosomal subunits requires ........ ? Ran.
What is used to export most of the mRNA outside the nuceloplasm? A mRNA exporter. (NOT RAN)
How many subunits contains the mRNA exporter? What are they? 2. NXF1 and NXT1. they are TWO DIFFERENT proteins.
What are mRNPs? messenger Ribonucloprotein particles.
NXF1 and NFT1 form __________ that bind to ___________ 1) dimers 2) mRNPs.
the NXF1 and NXT1 of mRNA exporter, once bound to mRNPs, can interact with the ____________ and go through the NPC. FG repeats
Transport of the the mRNP through the NPC is Ran-dependant or independent? independant.
What is DBP5? where is it located? It is an RNA helicase that is linked to the cytoplasmic filaments.
What is the main role of DBP5 as mRNA is exported out of the nucleoplasm? it destabilizes RNA-RNA interactions and RNA-protein interactions. NFX1 and NFT1 are ejected from the mRNA.
Do some of the SR proteins remain on the mRNA after splicing? Yes. They are also used in mRNA export.
What are Exon-junction complex proteins? Give an example. 1) They are proteins that remain on mRNA after siplicing and they are located at the junction of two exons. 2) REF.
What end of mRNA goes first through NPC during mRNA export? Why? The 5' end, because the 5' end GAP targets the mRNA through the NPC
Are Poly(A) binding proteins required for mRNA export? Yes.
What are the important things that have to stay on mRNA after its passage through NPC? CAP binding complex, NXT1 and NXF1 and PABP (PABPll is replaced by PABPl)
After mRNA goes through the NPC, PABPll is replaced by...? The CAP is replaced by ...? NFX1, NFT1 and REF are....? This whole step is called...? 1) PABPl 2) a translation initiation factor 3) released. 4) remodeling.
When pre-MRNA are not spliced correctly, can they be exported into the cytoplasm? Why? No, because there is a mechanism into the NPC that recognizes the splicing factors that are still on the mRNA and blocks it passage through the NPC.
How many types of functional RNAs are involved in protein synthesis? 3
What is the role of messenger RNA ? It carries the genetic information in the form of codons.
What is a codon? a series of three adjacent nucleotides that determine the insertion of a precise amino acid in the polypetide chain.
What is the role of tRNA? What in it structures allows it to do so? They decipher the codons on the mRNA. They have a three-nucleotide anticodon that can base pair with a codon in mRNA.
An amino acid requires how many tRNAs to bind to it and carry it into a polypeptide chain? one or more
What is the role of rRNAs? They associate with proteins to form ribosomes.
One of the rRNAs catalyses the formation of a peptide bond between what and what? the N of the amino acid group and the carboxy terminal C on the growing polypeptide chain.
How do we call a triple base pairing between a tRNA and a codon of mRNA? A trimer.
In what direction is the mRNA read in the ribosome? From the 5' end to the 3' end.
Are tRNAs heterogeneous particles? Yes
Are tRNAs conserved? Not necessarily in term of there sequence, but they have a very conserved structure.
How many base paired stems and loop regions have tRNAS? 4 base paired stems and 3 loop regions.
What sequence is always present at the 3' end of tRNAs? CCA
in tRNAs, the acceptor stem interacts with codons or with amino acids? amino acids.
What kind of RNA molecule has the higher level of posttranscriptional modifications? tRNAs.
What is the effect of the high number of posttranscriptional modifications in tRNAs? Non standard Watson-Crick base pairing.
What is particular with inosine? It can make nonstandard base pairing with other nucleotides.
What is a charged tRNA? a tRNA that is linked to an amino acid at its acceptor stem
Can a tRNA that has not the "L" structure make the system of translation work? No.
How many different tRNAs are there in prokaryotes? And in eukaryotes? 30-40, 50-100.
True or false : more than one tRNA can bring a certain amino acid? True : there are only 20 amino acids bu 500-100 different tRNAs in eukaryotes (per example).
What is the role of amino acyl-tRNA synthetase? It links a tRNA to a specific amino acid.
Does the linkage of a aminoacyl-tRNA synthetase to its amino acid and to a tRNA requires ATP? Yes.
In an aminoacyl-tRNA, the esther bond is formed between the aminoacid and which part of the tRNA? The CCA at its 3' end.
How many aminoacyl tRNA synthetase are there? 20
How could you explain the fact that there are many different tRNAs that can read a same sequence and bring the same amino acid? They some of the bases in wobble position of tRNA can undergo nonstandard base pairing!
How many possible codons are there in genetic code? 3
Give the 3 characteristics of the genetic code 1) comma-less (no interruptions between codons) 2)overlapping (it can be read in 3 possible frames) and 3) degenerate (more than one codon can encode the same amino acid)
The start codon is usually what sequence of amino acids? AUG (codon for methionine)
How many codons function as stop codons? What are they? 3 : UAA, UAG, UGA
How many additional small rRNAs contains the large subunit in prokaryote's ribosomes? And in eukaryote's? 1. 2.
How many proteins contain the large subunit of the ribosome? Between 31 (prokaryotes) and 50 (eukaryotes).
How many proteins contain the small subunit of the ribosome? Between 21 (prokaryotes) and 33(eukaryotes).
decoding the nucleotide sequence in mRNA requires...? tRNAs and aminoacyl-tRNA synthetase
an aminoacyl-tRNA synthetase can recognize how many different amino acids? ONLY 1!!
How many sites are there in the catalytic region of a ribosome? 3 (E=exist P=peptidyl A=acceptor)
How many steps are there in translation ? 3 : initiation, elongation, termination
Describe the initiation step of translation Assembly of a ribosome complexed with an mRNA and an initiator tRNA charged with methionine.
how many types of tRNAs are there for the methionine codon? 2 : tRNAimet and tRNAmet.
tRNAimet (initiator) is special because...? It recognizes the start codon in translation.
tRNAmet is exclusively used for...? elongation of the polypeptide chain.
The initiator tRNA can only go in which site of ribosome? And the non-initiator one? peptidyl site. acceptor site.
When is the mRNA functional? When it gets out of the Nuclear Pore Complex
What is quicker? control of translation or control of transcription? translation.
Is the structure of the ribosome (large and small subunit) is conserved between prokaryotes and eukaryotes? Yes.
Why all tRNAs have a similar 3D structure? To interact properly with the ribosome.
Is the active site of the ribosome conserved? Yes
Explain the role of regulation in the ribosomes structure. The two subunits of the ribosome are kept apart by a number of proteins : this is regulation. We want a regulated assembly of the ribosome onto the mRNA so that we get a regulated translation of that mRNA. :)
To be accepted into an acceptor site of the ribosome, tRNAs have to be...? charge (linked to its amino acid)
Into the cytoplasm, tRNAs are bound to...? initiation factors for translation.
which step contains an irreversible step in its initiation? Translation of mRNA (when a tRNA are bound to the good substrate, YOU LOCK IT.
both subunits of the ribosome contain a major rRNA or there is only one major rRNA per ribosome? Each subunit contains a major rRNA molecule.
what thing have in common the three steps of translation? there is an energy requiring step which results into a conformational shift into the ribosomes that allows to backtrack. (this involves GTP to GDP hydrolysis).
In eukaryotic translation, what is initiation? Assembly of a ribosome complexed with an mRNA and an initiator tRNA charged with methionine.
In eukaryotic translation, what is Elongation? Stepwise addition of amino acids to the polypeptide chain.
In eukaryotic translation, what is termination? Release of the completed polypeptide and of the ribosome, disassembly of the ribosome.
what does eIF mean? eukaryotic initiation factor.
Thanks to which initiation factors are kept apart the two subunits of the ribosome? eIF1, eIF3 and eIF 1A
What complex do we have to form before translation initiation? the 43S pre initiation complex.
What composes the eIF2 ternary complex? an initiator tRNA bound do methionine and to eIF2, which is bound to GTP.
What forms the 43S preinitiation complex? The 40S subunit of the ribosome (bound to eIF 1, eIF3 and eIF1A) and the tRNA (bound to methionine and to eIF2 linked to GTP).
When is eIF4 complex added to the 5' end of the mRNA? When mRNA is exported out of the nucleus. (mRNP remodelling)
In translation initiation, which PABP is present on the poly(A) tail? PABPl
In translation initiation, what composes the eIF4 complex? eIF4 bound to eIF4G (that will bind to the PABPl) and bound to eIF4A (which has RNA helicase activity)
what is the effect of eIF4A RNA helices? It unwinds the secondary structure and the 5' end of mRNA.
Describe the role of each eukaryotic initiation factors in activation of the mRNA before translation. eIF4A has RNA helicase activity, eIF4G binds to the PABPl (which links the 3' end to the 5' end), eIFB is a stimulator for RNA helicase, eIF4E bounds to the 5' end of mRNA
What positions the AUG initiation codon for pairing with the tRNAi in bacteria? The Shine-Dalgamo box.
What positions the AUG initiation codon for pairing with the tRNAi in eukaryotes? a relatively conserved sequence ACCAUGG recognizes the start site (AUG) KOZAK.
In RNA translation initiation, the activated mRNA forms a complex with which subunit of the ribosome? The small.
once the activated mRNA forms a complex with the small subunit of the ribosome, what other steps complete initiation of translation? 1) recruitment of the large ribosomal subunit and of the eIF5B-GTPase. 2)displacement of the eIF that were present on the mRNA 3)hydrolysis of the eIF5B-bound GTP and release of eIF5B and eIFA1
Can a single mRNA be translated by more than one ribosomes? Yes. It can be translated by multiple ribosomes.
In the ribosome, the peptidyl site is where we form...? The peptide bond.
In the ribosome, the A site is where the _______________ come in. aminoacyl-tRNAs.
The E site is where the ________ exit. tRNAs.
Once a tRNA is associated with its amino acid (charged tRNA), it is associated into the cytoplasm with ____________ ? EF1alpha (elongation factor 1 alpha)
What reaction occurs once the corresponding amino acyl-tRNA bins to a codon of the mRNA? Its EF1apha-GTP is hydrolyzed to a GDP
in translation, the peptidyltransferase reaction is catalyzed by what? The large ribosomal RNA.
What is ribosome translocation? It is when the mRNA is moved along on the distance of one codon. This process requires energy from EF2-GTP hydrolysis.
Are there sequences on the mRNA that are STOP codons? Yes.
In the translation termination, what is recruited instead of an amino acyl-tRNA? eRF1 and eRF3-GTP, (which are proteins, not RNAs)
what does eRF means? eukaryotic release factor
eRF3-GTP acts with eRF1 to promote what? cleavage of the peptidyl-tRNA.
At the end of translation, what is the role of ABCE1? It forms a complex to separate the two subunits of the ribosome and to release the translated mRNA.
what are the two factors that keep the two subunits of the ribosome apart in a regulated manner? eIF6 (on the large subunit) and eIF3 (on the small subunit)
What is the normal rate of elongation? 30-60 s for synthesis of a typical protein molecule.
What are the three cytoplasmic mechanisms of post-transcriptional control of gene expression. 1. Translation regulation 2. RNA degrading 3. mRNA localization (that causes anterior posterior polarity by bringing mRNAs where they are needed in embryonic development).
In the unfertilized egg, many mRNAs are stored with...? short poly(a) tail, which makes them translationally silent.
Why do we have to keep certain mRNAs silent in the unfertilized egg? Because there are certain genes that you don't want to express now!
Why is there a huge amount of mRNAs in the oocyte? Because the oocyte has to supply the embryo.
Why is the oocyte so huge? Because it contains a huge amount of mRNAs.
Before translation, mRNAs are linked to what proteins? eIF4 (5' end) and poly(A) binding proteins (3' end)
How do you form circular RNA before translation? The eIF4 (5' end) interacts with the PABP1 (3, end)
Why is it advantageous for mRNA to have a circular shape before translation? It enhances the concentration of translation factors in the vicinity so that you get higher translation of that molecule.
If you want that the mRNAs in the oocyte stay inactive until there is a signal that allow their activation, what do you do? You express a protein called Maskin.
Why do inactive mRNA have short poly(A) tails? Because they don't have enough PABP1 to interact with the translation machinery.
In the oocyte's mRNAs, there is an upstream region of the poly(A) tail that binds to a protein called...? CPEB
What happens in the oocyte when CPEB is NOT phosphorylated? it binds to Maskin (a protein) which binds to eIF4E and blocks it from binding to eIF4G : no translation.
CPEB binds to a region of mRNA which is __________ ? U-rich.
When Maskin is bound to eIF4E and to CPEB, the mRNA is almost ready for translation, because...? it is a complete RNA molecule with everything loaded in terms of translation initiation.
:In the circular form of eukaryotic mRNA (before translation) what happens when we dump the Maskin protein? CPEB loses contact of eiF4 and the new PABPl can now lengthen the poly(A) tail.
How can we do to remove the Maskin protein from the circular form of RNA (before translation)? We phosphorylate the CPEB.
before translation, when Maskin is released, what allows the lengthening of the poly(A) tail? CPEB binds to CPSF, which binds to Poly(a) polymerase (PAP), which lengthen the poly(A) tail.
Why is Iron important in the cell? It acts as a cofactor in many enzymes.
Why do we not want to have too much Iron in the cell? Because at high concentrations, it can damage DNA.
The ferritin mRNA encodes a protein that...? binds iron ions.
When Iron concentration is high, should we express ferritin? Yes.
When iron concentration is high, explain how the cell can translate more ferritin mRNA. High concentrations of iron make IRE-BP protein inactive. When it is inactive, it cannot bind to IREs (secondary structures at the 5' end of the ferritin mRNA) and the mRNA is translated.
When iron concentration is low, explain how the cell can reduce ferritin mRNA translation? Low iron concentrations make IRE-BP protein active. When it is active, it can bind to IREs, which block ferritin mRNA from being translated.
How exactly does the binding of IRE-BP proteins to IREs blocks ferritin mRNA translation? IRE-BP interferes with scanning of the methionine in that gene.
In the ferritin mRNA, where are the stem-loops to which IRE-BP bind? At the 5' end.
In the TfR mRNA, where are the step-loops to which IRE-BP bind? Near the 3' end.
What is the TfR mRNA? It is a receptor that regulates iron import in the cell.
What happens with TfR mRNA when there is a high iron concentration? You don't want to import iron anymore : the IRE-BP protein is inactive and the TfR is easily degraded.
What happens with TfR mRNA when there is a low iron concentration? You want to import more iron!! : The IRE-BP protein is active and it binds to the stem-loops (near the 3' end) which prevent the TfR mRNA from degradation.
What is the major characteristic of the IREs stem loops on the TfT mRNA and on the ferritin mRNA? They comport an A-U rich regions.
How many pathways are there for regulation by RNA degradation? 3 : decapping pathway, deadenylation-dependent pathway and endonucleolytic pathway.
Briefly describe the decapping pathway of regulation by RNA degradation. Decapping enzymes decap at the 5'end, which destabilizes the whole molecule and allows poly(a) tail shortening and exonucleolytic decay from the 5'end to the 3' end.
Briefly describe the deadenylation-dependent pathway of regulation by RNA degradation. The poly(A) tail of the mRNA is shortened by en enzyme and then a) the product can be decapped as in the decapping pathway or b) decayed from 3' end to 5' end by exonucleolytic decay.
Briefly describe the endonucleolytic pathway of regulation by RNA degradation. There is an endonucleolytic cleavage of the mRNA, which is then decayed from 3' end to 5' end by exonucleolytic decay.
What complex of enzyme is responsible of the exonucleolytic decay? exosome.
Regulated degradation is a mechanism used to ... silent expression of certain genes.
What are regulatory RNAs? Small RNAs (about 21 nts) that base pair with 3' UTR sequences of a specific mRNA.
What are the two types of regulatory RNAs? miRNAs and siRNAs.
do regulatory RNAs code for proteins? No.
What are miRNAs? micro RNAs. They are encoded by DNA and they are one of the two types of regulatory RNAs.
What are siRNAs? They are silencing RNAs. They are not encoded by genome's DNA and they are produced from double-stranded RNA, which never occurs in the cell EXCEPT in viral infection.
True or false : we have develop the mechanism involving miRNAs through viral infections. False : siRNAs.
miRNAs do/do not (?) base pair precisely with their target RNAs and __________ their translation. 1) do not 2) repress.
siRNAs do/do not (?) base pair precisely with their target RNAs and induce their ___________/_________ 1) do 2) cleavage/degradation.
miRNAs form a stem-loop structure : true or false? TRUE
After forming a stem-loop structure, what happens with miRNAs? They form a double-stranded RNA, which is never seen in the cell.
When one strand of the double-stranded mi/siRNAinteracts with the Dicer complex, what happens? It separate the two strands of the double-stranded RNA and incorporate those in another complex called RISC.
What does RISC mean? RNA-induced silencing complex.
After the ligation of a single-stranded miRNA with the RISC complex, what happens? The single-stranded miRNA finds a certain RNA message with which is imprecisely base pair.
What happens when single-stranded miRNA IMPRECISELY base pair with a certain RNA sequence? It blocks translation of this mRNA by an unknown mechanism.
siRNAs are recent or ancient? ancient, they would have appear before the animal/plants split.
After the ligation of a single-stranded siRNA with the RISC complex, what happens? The single-stranded siena precisely base pair with a certain RNA sequence and endonucleases contained in the RISC complex degrade this thing.
True or false : miRNA-RISC complex represses translation and siena-RISC complex causes the endonuclease from the RISC complex to degrade mRNA. True.
Created by: Julienrousseau
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