MCB EXAM 2
Upstream & Downstream
+1 position
Why is that important from the human health point of view?
- Antibiotics: inhibit a key function in bacteria without affecting cellular function in humans - Penicillin- cell wall synthesis -Ribosome- targeted antibiotics
mRNA - Processing involves:
- Capping - Polyadenylation (addition of ~200 A at the 3' end) - Transcriptional termination (brought about by Polyadenylation) - Splicing - nuclear Export
What are the two stages at which translational fidelity is determined & ensured?
- Charging of tRNA by aminoacyl tRNA synthesis - Pairing of anticodon in tRNA with the codon in mRNA
tRNA - Processing involves:
- Cleavage (@5' and 3' - the 5' processing is exerted by a RIBOZYME [RNase P]) - What's a Ribozyme? - Addition of a CCA sequence at the 3' end - Base modification - Loading of an amino acid at the 3' end
Activators are MODULAR proteins (2 DOMAINS) - what are those two domains? Why is modularity important?
- DNA-binding domain (DBD) - recognizes specific DNA sequences; does not need to unwind DNA to recognize - Activation domain - protein to protein interactions (even in the absence of DNA-binding domain); modifies chromatin structure - Modularity is essential in allowing combinatorial diversity
What's the main goal of translational initiation?
- Dissociate the ribosomes and keep them from coming back together ahead of time - Prevent tRNAs from joining the A site ahead of time - Bring together the tRNA, mRNA, small ribosomal subunit, and detect correct start codon - Allow large ribosomal subunit to join the small one and allow entry of tRNAs into A site
Differences between prokaryotes and eukaryotes at different stages of the process (enzymes; co-factors; promoters)
- General - involved in transcription from all promoters (basic transcription machinery) - Specific - bind to DNA sequences that control expression of individual genes (regulation of transcription)
Transcription factors: General vs. Specific - What's the difference? What do they do?
- General - involved in transcription from all promoters (basic transcription machinery) - Specific - bind to DNA sequences that control expression of individual genes (regulation of transcription)
What's the role of Aminoacyl-tRNA-synthetases?
- Maintain fidelity - Put the right amino acid in the right tRNA - charging of the tRNA
Players involved in translation:
- Messenger RNA (mRNA) - Transfer RNA (tRNA) & Aminoacyl-tRNA synthetases - Ribosomal RNA & Ribosomes - Protein Factors (Initiation, Elongation, & Termination Factors)
Mechanisms of mRNA Degradation
- Nuclear - Non-sense mediated mRNA decay - Cytoplasmic - > Decapping pathway >Deadenylation-dependent pathway >Endonucleolytic pathway
Factors that activate the mRNA - what do all of them do?
- Poly-Adenyl Binding Protein (PABP) - associated with the poly(A) tail at the 3' end of the mRNA; stimulates translation by interacting with eIF4G - eIF4G (remember, G=Grouping) - binds to PABP; one of the initiation factors (other is EIF4E); in association with eIF4A and eIF4B along with initiation factor eIF4E, it brings the mRNA to the 40S ribosomal subunit, with eIF4G interacting with eIF3 - eIF4E - recognizes the 5' cap of the mRNA and forms a complex with eIF4A and eIF4G - eIF4A and eIF4B - in association with initiation factors eIF4E and eIF4G, they bring the mRNA to the 40S ribosomal subunit, with eIF4G interacting with eIF3
How is DNA condensation regulated?
- Post-translational modifications of HISTONES - Nucleosome Remodeling Factors - miRNAs - Chemical modifications of DNA - DNA METHYLATION
Template
- Prokaryotic RNA Polymerase - circular DNA not organized in nucleosomes - Eukaryotic RNA Polymerase - linear DNA organized in nucleosomes
3 RNA Polymerases:
- Prokaryotic RNA Polymerase - synthesizes all prokaryotic RNAs -RNA Polymerase I - synthesizes ribosomal RNAs (28S, 18S, and 5.8S) -RNA Polymerase II - synthesizes protein-coding RNAs (mRNA) -RNA Polymerase III - synthesizes smallest ribosomal RNA (5S), transfer RNA, and some of the small RNAs involved in splicing and protein transport
How is the start codon selected in prokaryotic cells? What's the Shine-Dalgarno sequence? Key to remember: "A Gorgeous Girl Always Gives Guys Ulcers"
- Prokaryotic don't have a cap and are polycistronic - Have the Shine-Dalgarno sequence: Initiation codons in bacterial mRNA; a ribosomal binding site in the mRNA, generally located 8 basepairs upstream of the start codon AUG. The six-base consensus sequence is AGGAGG. Lacated very short distances from the start codon
DNA Sequences involved // Protein factors that recognize each one of them
- Promoter // General Transcription Factors - Cis-acting sequences (proximal control elements) // Specific Transcription Factors or ACTIVATORS - Enhancers // Specific Transcription Factors or Activators - Silencers // Repressors
Types of RNA-Polymerases & specific functions
- RNA-Polymerase I - synthesizes ribosomal RNAs (28S, 18S, and 5.8S) - RNA-Polymerase II - synthesizes protein-coding RNAs (mRNA) - RNA-Polymerase III - synthesizes smallest ribosomal RNA (5S), transfer RNA, and some of the small RNAs involved in splicing and protein transport
Regulation is exerted at four different levels:
- Sequence Elements that regulate binding/initiation - Regulating DNA condensation (packing) - Regulating Processing (of the transcript) - Regulating transcript stability
How can you regulate transcript levels? Four mechanisms
- Sequence elements that regulate binding/initiation (core promoter, cis-acting elements, enhancers, silencers; each recognized by specific protein factors) - Regulating DNA packing/condensation (Histone Acetylation/De-acetylation, Histone-methylation, DNA methylation) - Regulating processing of the transcript (splicing, nuclear export) - Regulating stability of the transcript (degradation)
Binding stage: Factors involved and what they do
- TFIID(TATA-Binding Protein[TBP]+TATA-Associated Proteins [TAPs])(how does TBP mark the region in the DNA where the promoter is located?) -TFIIB (B=bridges interaction with RNA-Pol II) - RNA-Pol II+TFIIF -TFIIE+TFIIH (TFIIH contains 2 different activities, HELICASE [exerted by the Xeroderma pigmentosum proteins XPB and XPD] and KINASE activity and is required to unwind the DNA to start transcription and to phosphorylate the Carboxyl-Terminal Domain or CTD in RNA-PolII) -Phosphorylation of the CTD of RNA-PolII releases the RNA-PolII from the pre-initiation complex and allows active transcription to start
How is transcription termination achieved in prokaryotes?
- Termination Sequence - symmetrical inverted repeat of a GC-rich sequence followed by four or more A residues; results in the formation of a stable stem-loop structure - Rho - ATP-dependent unwinding enzyme (moves along the newly formed RNA molecule towards its 3' end and unwinds it from the DNA template)
Whats an Open Reading Frame
- Three nucleotides encode from one amino acid - (4)^3 = 64 different potential combinations for only 20 amino acids - Redundant Degenerate - Extreme care must be placed on selecting the right ORFs otherwise may end up with something that is completely unreadable or a "nonsense."
Pay special attention to the following methods of translational regulation:
- miRNAs - Phosphorylation of eIF2 and eIF2B - Phosphorylation of 4E-BPs
Kozak sequence
- plays a major role in the initiation of the translation process - a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG - (A/G) AUGG translation (A/G: -3) (AUG: +1) (G: +4)
What's the direction of transcription?
5' to 3' direction
eIF2-GTP - role in translational initiation
A GTPase; the one that senses the recognition of the start site by the tRNA
By the way, what's the PROTEASOME?
A large protease complex that degrades proteins tagged by ubiquitin.
Polysomes vs. Monosomes - why does it matter? What's a polysome?
A polysome is also known as a polyribosome - it is the group of ribosomes bound to an mRNA molecule that translate it simultaneously; spaced at intervals of about 100-200 nucleotides. mRNAs are usually transcribed in this manner, where each ribosome within the group functions independently to synthesize a separate polypeptide chain.
What is feedback inhibition?
A type of allosteric regulation in which the product of a metabolic pathway inhibits the activity of an enzyme involved in its synthesis.
Besides keeping the small ribosomal subunit away from the large, what else does eIF3 do?
Allows the interaction with the mRNA - specifically with eIF4G
What's the main effect of lipidation on proteins?
Allows them to associate with membranes.
What limits the range of activity of enhancers?
Although DNA looping allow enhancers to act at a considerable distance from promoters, the activity of any given enhancer is specific for the promoter of its appropriate target gene. Enhancers, like promoters, function by binding transcription factors that then regulate RNA polymerase. Specificity- Maintained by Insulators or Barrier elements
What's a phosphatase?
An enzyme that reverses the action of protein kinases by removing phosphate groups from phosphorylated amino acid residues.
How is GTP-loaded (or bound) eEF1alpha recycled? What's the name of the GTP-Exchange Factor for this process?
As elongation continues, the eEF1α that is released from the ribosome bound to GDP must be reconverted to its GTP form. The GTP-Exchange Factor is eEF1βγ, which binds to the eEF1α/GDP complex and promotes the exchange of bound GDP for GTP, resulting in the regeneration of eEF1α/GTP that is now ready to escort a new aminoacyl tRNA to the A site of the ribosome - beginning a new cycle of elongation.
Levels of transcription
BASAL (+); Stimulated by Cis-acting sequences bound by Activators (up to +++); Stimulated by Enhancers bound by Activators (up to ++++++)
Stages during the process of transcription
Binding, Initiation, Elongation and Termination
What's the role of eEF1alpha?
Brings enzyme; GTPase enhances translational fidelity
The vast majority of proteins that are translocated into the ER are
CO-TRANSLATIONALLY N-Glycosylated.
What is capping? When does it happen? What are the enzymes involved in the process bound to?
Capping involves 7-methylguanosine serving as the "cap" that ensures mRNA stability, prevents rapid degradation of the RNA which will avoid the inhibition of translation, allows binding of initiation factors needed for translation, and helps regulate nuclear export of the mRNA; the process starts soon after transcriptional initiation; the enzymes involved are bound to the phosphorylated CTD of RNA-Pol II following initiation of transcription
How is the activity of Activators regulated?
Cellular levels, cellular localization, post-translational modification
What do Chaperonins do? How is that different from what chaperons do?
Chaperons help maintain the protein in an open conformation, therefore preventing aggregation and misfolding. Chaperonins provide an "isolated" environment for proteins to fold in the absence of other interfering factors.
MAPPING OF PROMOTERS -
Cis acting sequences, core promoters, enhancers
Where are cis-acting sequences located?
Close to the promoter (100-200 bp away); can be located upstream or downstream from the core promoter
Genomic imprinting - what is it? How is it achieve?
Controls the expression of some genes involved in the development of mammalian embryos; imprinted genes are dependable on the expression of whether they are inherited from the mother or from the father
DNA-Methyl transferases - what do they do? what's their effect in DNA packaging?
Controls transcription; DNA is methylated specifically at the cytosines that precede guanines in the DNA chain (CpG dinucleotides), and this methylation is correlated with transcriptional repression; associated with transcriptional repression of some genes, in concert with alterations in chromatin structure
Role of the promoter - what does it do? Why is it essential?
DNA sequence to which RNA-Pol binds to initiate transcription of a gene; directs RNA Polymerase to the template; dictates which of the two DNA strands will serve as the template; positions RNA Polymerase over the initiation site
Termination
Different for every RNA-Pol; for RNA-PolII termination occurs after PolyAdenylation and is triggered by it
Histone Methyl-Transferases (H-MTs) - How do they affect DNA packing? why? (remember the challenge question)
Does NOT affect charge - Instead, it provides a binding surface for various activators or repressors; Methylation Histones can lead to both increased transcription or decreased transcription
Transferrin Receptor mRNA
Exemplifies mRNA degradation regulated by presence of a factor that protects the mRNA from the endonucleolytic pathway
What is Huntington's disease associated to?
Expansion of Poly-Glutamine (Q) repeats in the sequence of Huntingtin. 33 Qs or less = normal protein; 36 Qs or more = mutated Huntingtin - the protein is mislocalized and misfolded - leads to neuron death.
Histone ACETYLATION & DE-ACETYLATION - Histone Acetyl-Transferases (HATs) & Histone De-Acetylases (HDACs) - How do they affect DNA packing? why?
HATs are characteristic of actively transcribed chromatin (alters CHARGE of histones thus affecting their ability to form a tight closed structure with DNA) whereas HDACs are associated with inactive chromatin that cannot be transcribed; transcriptional activators and repressors are associated with coactivators and corepressors, which have HAT and HDAC activities, respectively
What's the name of the different regions in the ribosome that are involved in tRNA binding?
Has 3 sites = P (peptidyl), A (aminoacyl), and E (exit) sites
Why is rRNA important?
Heart of the ribosome; RNA; recognition of correct codon-anticodon pairing in the decoding center, like peptidul transferase activity, is principally based on rRNA activity
What kind of physiological conditions trigger an upregulation in the synthesis of chaperons?
Heat shock and other stress conditions. Hence their name "Heat Shock Proteins."
Template for transcription in eukaryotes - organized in NUCLEOSOMES - why is that important?
Highly condensed DNA is transcriptionally inactive; having it organized as individual nucleosomes will allow for transcription to happen as it is the basic structural unit of chromatin
Initiation
Incorporation of first 2 nucleotides in nascent RNA chain
How can miRNAs affect DNA packing? What's the ultimate effect? What's the name of the enzyme that mediates that effect? Does the miRNA have to match the RNA precisely or are mismatches allowed?
Induces histone modifications that lead to chromatin condensation and the formation of heterochromatin, repressing transcription; the enzyme is the RISC complex; mismatches are allowed
What's involved in creating a new peptide bond? What's released during the process?
Insertion of a correct aminoacyl tRNA into the A site triggers a conformational change that induces the hydrolysis of GTP bound to eEF1α and release of the elongation factor bound to GDP. Once eEF1α has left the ribosome, a peptide bond can be formed between initiator methionyl tRNA at the P site and the second aminoacyl tRNA at the A site. This reaction is catalyzed by the large ribosomal subunit, with the rRNA playing a critical role.
Combinatorial diversity - what is it? why is it important?
It allows for a wide variety of genes in cells (in part, mediated by transcription factors) and provides insight as to how transcription factors shape gene expression
Post-translational modifications
It can be either REVERSIBLE or IRREVERSIBLE (keep in mind: Post-translational modifications ALMOST ALWAYS ALTER THE SHAPE/CONFORMATION of the protein and play an important role in folding)
What's the role of chaperons?
It facilitates the correct folding or assembly of other proteins, preventing protein aggregation.
O-Glycosylation
It involves the addition of one sugar at a time.
N-Glycosylation
It involves the transfer of a pre-formed chain made of 14 different sugars.
What's a GPI anchor? How is it added to the protein? What's the final topology and location of ALL GPI anchored proteins?
It is a glycolipid containing phosphatidylinositol that anchors proteins to the external face of the plasma membrane. The oligosaccharide portions of GPI anchors are attached to the terminal carboxyl group of polypeptide chains. The fatty acid chains of the GPI anchor mediate its attachment to the plasma membrane.
Phosphorylation
It is the most transient post-translational modification (phosphate groups added to proteins can be easily removed), and therefore constitutes the best example of a REVERSIBLE post-translational modification.
met-tRNA - why is it always the first amino acid used to start translation?
It is the only one with the ability to bind to the P site directly
Protein cleavage
It is the only truly fully IRREVERSIBLE post-translational modification (once the protein is broken, it can't be put together again). It is a frequent event during the targeting of proteins to the Secretory Pathway (translocation into the ER).
Role of P-TEFb?
It neutralizes the negative action of NELF and DSIF by binding to RNA-Pol II, releasing NELF and phosphorylating DSIF that further neutralizes its inhibitory activity on RNA-Pol II. Its positive action drives RNA-Pol II into the highly processive elongation stage of RNA transcription
Protein glycosylation
It occurs either at Asparagine (Asn/N)[N-Glycosylation] residues OR at Serine (Ser/S) or Threonine (Thr/T) residues [O-Glycosylation as in this case the functional group where the sugar is attached is the OH group, leaving an -O- link].
Role of Mediator
It stimulates transcription of eukaryotic protein-coding genes and allows them to respond to gene-specific regulatory factors.
If you wanted to specifically inhibit the translation of one specific mRNA, what would be the best approach?
It would be RNA interference
What's a KINASE? How many types are there?
KINASE: protein that couples phosphate groups to its target proteins. There are two main types: Serine/Threonine kinases and Tyrosine kinases (depending upon their ability to phosphorylate Serine/Threonine residues or Tyrosine residues).
What's the template used for transcription?
Linear DNA organized in nucleosomes
rRNA processing
Main processing event: Cleavage (keep in mind which rRNAs are encoded in the same transcriptional unit and which rRNA is encoded in a separate gene in a different chromosomal location)
What is Alzheimer's disease associated to?
Misfolded Amyloid Precursor Protein (APP).
What is Osteogenesis Imperfecta associated to?
Misfolded Collagen I
How many different types of lipid modification are there? In reference to the primary sequence of the protein, where does each one of them occur? (think in terms of N-terminus, C-terminus, or middle, also keep in mind what amino acid residue is used in each case).
Myristoylation - N-terminal of glycine residue Farnesylation - C-terminal of cysteine residue Palmitoylation - middle of cysteine residue
Role of DSIF and NELF? Why is it important to halt the RNA-PolII within about 50 nucleotides of the transcriptional start site? What is achieved with that?
NELF and DSIF produce a dramatic slow-down in RNA-Pol II transcription, resulting in a complete pause right around the time when capping takes place. Capping is a process that occurs early during transcription and essential to ensure the survival of the transcript which would otherwise be rapidly degraded by exonucleases
Is the binding of small molecules to a protein a type of post-translational modification?
No (because to be consider a post-translational modification the molecule added must be covalently associated to the protein), but the binding of small molecules to a protein can exert dramatic changes in protein shape and function.
Requirements for transcription - are primers required?
No Primer required = Primase if an RNA-Polymerase
Is the active form of a protein always the phosphorylated one?
No because either the phosphorylated or dephosphorylated protein can be active.
Regulation
Occurs mainly at the Binding/Initiation stage (once transcription is initiated, it usually goes to completion
What's the role of PDI (Protein Disulfide Isomerase)? How does it contribute to protein folding? Does it play a role in the folding of cytosolic proteins? Where is PDI located, i.e. in which cellular organelle?
PDI catalyzes the formation and breakage of disulfide (S-S) linkages. It promotes rapid exchanges between paired disulfides, thereby allowing the protein to attain the pattern of disulfide bonds that is compatible with its stably folded conformation. It does play a role in the folding of cytosolic proteins because the cytosol contains reducing agents that maintain cysteine residues in their reduced form, thereby preventing the formation of disulfide (S-S) linkages. It is located in the endoplasmic reticulum.
Evidence that DNA packing (condensation) is related to Transcriptional Regulation (degree of condensation dictates transcriptability)
Packaging of eukaryotic DNA into chromatin limits its availability as a template for transcription, so chromatin structure is a critical aspect of gene expression in eukaryotic cells. Rather than being present within the nucleus as naked DNA, the DNA of all eukaryotic cells is tightly bound to histones. The basic structural unit of chromatin is the nucleosome. The chromatin is further condensed by being coiled into higher-order structures organized into large loops of DNA.
Differences between translational initiation in prokaryotes and eukaryotes
Prokaryotes: - IF3 prevents two these two things to get back together - Factor One (A side) preventing rRNA from entering small subunit mediated by the Shine Delgarno sequence - IF2 ( bound to GTP) sensor; bringing mRNA will also bring the tRNA -Forms methionyl - ONLY acess to the P side Nformyl -methionyl tRNA the rest are placed in the A side - GTP event Eukaryotes: - dissociate by eIF3 - prevent tRNA to the A side 1A (in prokaryotes is just 1) - bring everything together recognizes the cap - cap binding protein eIF4 - Factor four G eIF4G - Group everything together - eIF4A + eIF4B need help from one another (scanning secondary structure) - tRNA directly methionyl Aminiacyl tRNA - start codon: scanning your message RNA
Differences between eukaryotic mRNA and prokaryotic mRNA
Prokaryotic do NOT have a Cap - Shine-Dalgarno sequence - Located at very short distance from the start codon - Polycistronic- code for more than one protein (multiple protein) Eukaryotic need a Cap -5'end can be far away from the start codon - Kozak consensus sequence charge allows the recognition - Monocistronic -encodes for a single protein (regulating every single component) allow more flexibility
siRNA
Provides a mechanism to target specific mRNAs to be degraded by the endonucleolytic pathway - mediated by RISC
What's the role of eEF2?
Push ribosome to the next position; translocation: 3 steps driven by elongation factor eEF2
Main enzyme involved in transcription?
RNA polymerase
Elongation - once the RNA-PolII has disengaged from the pre-initiation complex (which contains XPB and XPD), how does RNA-PolII manage to open the dsDNA to continue reading the template?
RNA-Pol II is associated with Mediator proteins, as well as with the general transcription factors, at the promoter. The Mediator complex binds to the nonphosphorylated CTD of polymerase II and is released following phosphorylation of the CTD when transcription initiates. The phosphorylated CTD binds other proteins (elongation and processing factors) that facilitate transcriptional elongation and function in mRNA processing.
Why is conformation important?
Remember: the two most important determinants/regulators of protein function are shape (conformation) and cellular localization. The protein must have the right shape and be localized in the right cellular location to be able to perform its job.
How is the start codon selected in eukaryotic cells?
Ribosomes recognize most eukaryotic mRNAs by binding to the 7-methylguanosine cap at their 5' terminus and then scan downstream of the 5' cap until they encounter the initiation codon (usually AUG)
What is SUMO?
SUMO stands for Small Ubiquitin-like Modifier; hence, SUMO is a Ubiquitin-like protein that can also be post-translationally conjugated to other proteins in the same way as Ubiquitin.
IF2-GTP - role in the process
Sensor; bring mRNA will also bring the tRNA
snRNAs - what are they? what do they do? which ones are the ones that exhibit the ability to catalyze the first stage of splicing (formation of the lariat-like intermediate)?
Small nuclear RNAs; they are the RNA components of the spliceosome (U1, U2, U4, U5, and U6); they are complexed with 6 to 10 protein molecules to form small nuclear ribonucleoprotein particles (snRNPs), which play central roles in the splicing process; U2 and U6 are responsible for catalyzing the first stage of splicing Splicing is also an RNA-mediated reaction (driven by a Ribozyme)
How do proteins achieve their final conformation?
Spontaneously, all the info need is contained within the primary sequence of the protein.
What's the role of the 5' cap and the 3' poly(A) tail on translational initiation?
The 5' cap and 3' poly(A) tail ensure mRNA stability; 5' cap is required for translation whereas 3' poly(A) tail only enhances translation and is not necessarily needed
What's the chemical group used to link the amino acid residue to the tRNA? Why is that relevant?
The Carboxyl group; it determines the direction of protein synthesis, from Amino (which is free for the first residue incorporated) to Carboxyl end
What's wobble? Why is it important?
The ability to establish non-standard base pairings at the third position of the codon; its relevance is to allow a single tRNA to recognize different codons
What is phosphorylation?
The addition of a phosphate group to a molecule.
If you wanted to inhibit translation (all together, for all possible transcripts) which would be the best alternatives?
The best alternatives would be phosphorylating eIF2 or eIF2B by regulatory protein kinases or by eIF4E binding proteins (4E-BPs), which bind to eIF4E and block its interaction with eIF4G.
What is a signal transduction pathway?
The cascade of events triggered by a signal received at the plasma membrane - usually results in the transcription of a specific set of genes. Signal transduction pathways usually involve a series of phosphorylation and dephosphorylation events.
What are post-translational modifications?
The enzymatic processing of a polypeptide chain after translation from mRNA and after peptide bond formation has occurred.
Transesterification - what is it? When does it happen?
The formation of a new phosphodiester bond between nucleotides that were previously bound to other nucleotides; occurs in the joining of exons
How can protein-protein interactions regulate protein function?
The importance of these interactions is evident in many allosteric enzymes in which the binding of a regulatory molecule alters protein conformation by changing the interactions between subunits.
How is transcription by RNA-PolII different from that driven by other RNA-Pols?
The location of the core promoter varies and the general transcription factors involved are different; the processing of the transcripts generated is also different and the process by which termination is achieved is also different.
Mechanisms by which translational regulation can be achieved - Which ones are global and which ones are specific to one transcript?
The mechanisms are the binding of repressor proteins to specific mRNA sequences (specific to one transcript), the binding of proteins to specific sequences in the 3' untranslated regions of some mRNAs (specific to one transcript), modulation of the activity of initiation factors (global).
What is allosteric regulation?
The regulation of enzymes by small molecules that bind to a site distinct from the active site, changing the conformation and catalytic activity of the enzyme.
Bacterial transcription - What's the role of the sigma subunit of RNA-Pol? What is its eukaryotic equivalent?
The sigma subunit is required to identify the correct sites for transcription initiation; The eukaryotic equivalent is specific transcription factors (TBP, TAFs, TFIIB, TFIIF, TFIIE, and TFIIH)
What is the signal used for polyadenylation?
The signal is the polyadenylation signal [Poly(A) signal], consisting of the hexanucleotide AAUAAA in addition to upstream and downstream (G-U rich) elements
Why is the genetic code said to be DEGENERATE?
The third position is usually less restrictive (in terms of specificity) due to the Wobble base pairing effect
How many codons are there? Start codons? Stop codons?
There are 64 different codons; 1 start codon; 3 stop codons
Is there translational regulation in bacteria? Why?
There is translational regulation in bacteria because they contain the subunits need to have translation.
Why are TFIID and TFIIB and a few other general transcription factors left in place upon release of the RNA-PolII? What is achieved with that?
They act as a place holder once RNA-Pol II is done transcribing; transcription is achieved.
Silencers - are they similar to activators? How can they inhibit transcription? Can they affect the activity of activators?
They are in no way similar to activators as they activate transcription and silencers inhibit transcription; silencers recruit repressors which in turn recruits Histone De-Acetylases (HDACs) in order to inhibit transcription; yes
Glycosylation (addition of carbohydrates) and lipid addition (lipidation)
They are usually final (not reverted under normal conditions) and therefore can be classified as SELDOM REVERTED (even though sugar chains usually undergo significant trimming and modification, and proteins can be de-lipidated under certain conditions).
How do Nucleosome remodeling factors act in combination with HATs, HDACs, H-MTs, and D-MTs?
They can be recruited to DNA in association with either transcriptional activators or repressors, and can alter the arrangement of nucleosomes to either stimulate or inhibit transcription
What's the main effect mediated by the conjugation of SUMO to a target protein?
They serve as markers for protein localization and as regulators of protein activity.
What are the roles of the cap and the polyadenyl tail?
They stabilize the mRNA and help regulate translation
Where are core promoters located?
Transcription with only the promoter induces only a basal level of transcription
Splicing - Stages involved, selection of the branching point, sequences that are always conserved at the 5' end (GU) and at the 3' end (AG) of the intron
U2 snRNP binds to the branch point by similar complementary base pairing between U2 snRNA and branch point sequences
What is Ubiquitin?
Ubiquitin is a small protein (only 76 amino acid residues long) that can be post-translationally conjugated to other proteins via iso-peptide links formed between the C-terminal glycine residue in Ubiquitin and the epsilon-amino group in Lysine residues located within the target protein to be Ubiquitinylated.
What's the main effect mediated by the conjugation of Ubiquitin to a target protein?
Ubiquitinylation usually targets the modified protein to be degraded by the PROTEASOME.
What do Nucleosome Remodeling Factors do?
Use energy derived from the hydrolysis of ATP to alter the contacts between DNA and histones; may act by inducing changes in the conformation of nucleosomes, affecting the ability of specific DNA sequences to interact with transcriptional regulatory proteins; can eject histones from the DNA, leaving a nucleosome-free region; can be recruited to DNA in association with either transcriptional activators or repressors, and can alter the arrangement of nucleosomes to either stimulate or inhibit transcription; Make sequences visible "push" nucleosomes to the side; Energy required-ATP consuming machine that pushes nucleosomes
Where are enhancers located?
Usually very distant from the promoter (100,000 bp away); upstream/downstream from gene; can be inverted, but still functional
What's the signal that triggers disassembly of the 48 S Pre-Initiation Complex?
When eIF2 hydrolyzes GTP to GDP, triggering a conformational change in eIF2 that results in the disruption of the 48S pre-initiation complex
What triggers GTP hydrolysis by IF2 in prokaryotes?
When the 50S ribosomal subunit associates with the complex containing the 30S ribosomal subunit, IF1, IF3, IF2 (bound to GTP), mRNA, and N-formylmethionyl tRNA, with IF2 specifically interacting with the initiator tRNA
Is the Ribosome a Ribozyme?
Yes because the catalytic peptidyl transferase activity that links amino acids together is performed by the ribosomal RNA
Are there any differences between eukaryotic and prokaryotic ribosomes?
Yes compositions are different Eukaryotic ribosome - 80S: small subunit 40S, large subunit 60S - 20S, 5.8S, and 5S present in the 60S - 18S present in the 40S subunit Prokaryotic ribosome - 70S: small subunit 30S, large subunit 50S - 16S only subunit in prokaryotes
Are there any diseases associated to defects in protein folding?
Yes. Osteogenesis Imperfecta (OI), Hungtinton's Disease, and Alzheimer's Disease are examples.
What are the factors that keep the small ribosomal subunit from binding the large ribosomal subunit?
elF3
What's the factor that activates the GTPase activity of eIF2?
elF5
Why does scanning require ATP hydrolysis?
it requires energy to do so
Following nuclear export
mRNA is Translated (in association with Ribosomes), and then decays - DEGRADATION
How is the mRNA read?
mRNAs contain Open Reading Frames (ORFs) and extreme care must be placed on selecting the right ORF; read in the 5' to 3' direction
What are the main residues that can be phosphorylated?
serine, threonine, tyrosine
Whats a codon?
the basic unit of genetic code; one of the 64 nucleotide triplets that code for an amino acid or stop sequence
