gene expression and regulation
how does the alternative splice choice mechanism occur?
the primary transcript can be used for the thyroid or brain or foot, and that's through a different cleave and polyadenylation that leads to different splicing and translation the only difference is the original transcript and how a hormone bound to the promotor region
what is transcription?
the process by which an enzyme system converts genetic information in dsDNA into an RNA strand with a complementary base sequence
how does protein kinase help in translation control? work with heme as an example
the protein kinase can add phosphate to change how the protein interacts with the kinase. for example, in low amounts of heme, the protein kinase will phosphorylate initiation factor 2 to inhibit the 40S initiation complex and work as a hemin controlled translation repressor
what are the common promotor motifs used in rna synthesis intiation?
TTGACA at -35 TATAAT at -10
how do we increase the increase of protein variety
alternative splicing mixed with poly A site choose increase the variety of proteins generated from the genomes of high eukaryotes by way of alternative transcript production
what enzyme catalyzes dna to rna ? what does it require? What direction is it build?
dna dependent RNA polymerase catalyzes transcription and this process requires a dna template, 4 ribonucleotide 5' triphosphates (atp, gtp, utp, ctp) and mg2+ rna polymerase builds RNA in the 5' to 3' direction by adding to the 3' OH end
what is required for RNA biosynthesis? how many strands are used as a template for a given region?
double-stranded DNA template DNA-dependent RNA polymerase (depends on DNA) 4 nucleotide triphosphates (atp, gtp, ctp, utp) mg++ for optimal activity and the polymerase working in 5' to 3' direction only 1 strand is used per template for a given region
how many nuclear RNA polymerases do eukaryotes have? What are they? how do they differ?
eukaryotes have 3 RNA polymerase 1, RNA polymerase 2, and RNA polymerase 3 they are all called by a specific promotor sequence for their function RNA polymerase 1 located in nucleus synthesizes pre-ribosomal RNA RNA polymerase II located in nucleoplasm synthesizes mRNA and ncRNA (will not code protein) and RNA polymerase III located in nucleoplasm synthesizes tRNA, 5S rRNA and ncRNA (will make 5S rRNA, and 45S RNA)
how does initiation of transcription occur? how about elongation?
intitiation occurs when RNA polymerase binds at promoter sequences (no promoters are required) the growing RNA strand temporarily base pairs with DNA template to forma short hybrid of RNA/DNA double helix
describe the initiation process of RNA polymerase II what apparatus is formed?
initiation begins when TATA box is recognized by the TATA box binding protein component of TFIID (this protein disassociates once RNA polymerase II binds) TFIIA is followed by TFIIB (seen in RNA polymerase II complex) and this forms a basal transcription apparatus the double helix then partially unwinds and this allows transcription to proceed
what does regulated gene expression bring about? define inducible define repressible how do they compare to each other
it can bring about increases or decreases in the amount of gene product inducible is the gene products increase in concentration under particular molecular circumstances repressible is the gene product decrease in concentration in response to a molecular signal induction is gene expression increase and repression is gene expression decrease
what factors help guide RNA polymerase II?
it is guided by TATA box at -25 on 5' end of start site but it also needs basal transcription factors
what is RNA polymerase I responsible for? What precursor does this rna contain? how does this differ from polymerase III?
it is responsible for the synthesis of a transcript called pre-ribosomal RNA pre-rRNA contains the precuroser for the 18S, 5.8S, and 28S rRNAs (45S RNA) polymerase III is responsible for the synthesis of tRNAs, the 5S rRNA and other ncRNAs and its Polymerase promoters are well characterized
what is RNA polymerase II responsible for? How many promoters can it recognize? what sequences do Polymerase II promoters have in common?
it is responsible for the synthesis of mRNAs and many ncRNAs, and can recognize 1000s of promotors has TATA box consensus sequence near -30 and a initiator seqeunce at +1
what is the splicing consensus sequence on an intron? what is located 20-50 nucleotides upstream of this
it is the base sequence on an intron that begins with GU and ends with AG that signals which introns should be spliced the branch point which is the location where nucleophilic attack can occur where the exons can combine after the intron is released
what genes does the lac operon have? What are these genes responsible in regards to expression? in the absence of lactose what happens to the operon?
lactose operon has genes for beta-galactosidase on gene (Z) ( for release of lactose hydrolyzing enzyme) Gene Y codes for galactoside permease (Y) (increases permeability for release of lactose into membrane) Gene A codes for the modification of toxic galactosidase to facilitate their removal lac operon is reversibly repressed
any hormones with a ligand-binding domain is a? when a hormone enters the cell what happens
ligand the hormone will bind the ligand binding domain which will release the inhibitor. that glucocorticoid complex that ligand bound to will move to the nucleus to activate the response element in the nucleus for the transcription of RNA
when lactose is high, glucose is high and cAMP is low what happens to the lac operon?
low transcription, because the repressor is still being bound, but some transcription will occur
when lactose is low, glucose is high and cAMP is low what happens to the lac operon?
no mRNA transcription because the cAP can not bind the activator protein or bind Lac Z
what is the most common intron in eukaryotes? What makes sites of splicing? What are some splicing apparatus components tehther to? why?
spliceosome introns GU at the 5' end and the AG at the 3' end mark the sites of splicing some splicing apparatus components are tethered to the CTD of RNA polymerase this is because splicing is tightly coordinated with transcription and will commence right after the polymerase II has finished synthesizing RNA
how does the core rna polymerase of ecoli compare to the holoenzyme for rna polymerase for ecoli?
the core enzyme is composed of all subunits except sigma subunit the holoenzyme has all subunits including the sigma subunit
how is rna polymerase transcription initiation regulated? compare these factors
with specificity factors, repressors and activators specificity factors alter the specificity of RNA polymerase for a given promotor or set of promotors repressors restrict access of RNA polymerase to the promotor activators enhance the RNA polymerase promotor interaction
what forms of RNA are degraded in the molecular process of gene expression
when the RNA is improperly processed it will be degraded while in the nucleus and outside the nucleus the mRNA will decay after a set amount of time
most eukaryotic genes are switched on how?
with the action of several transcriptional activators that contain a DNA binding domain and activation domain
describe the structure of rna polymerase of ecoli
2 alpha units that bind regulatory sequences 1 beta subunit that forms phosphodiester bonds one beta' subunit that binds DNA template 1 omega subunit that functions in conformation maintenance and recruitment of the beta' subunit one sigma70 that recognizes the promoter and initiates synthesis
how many subunits make up the RNA polymerase holoenzyme? What is a function of subunit sigma? How are the variants designated? What is its most predominant subunit form?
6 subunits sigma subunit directs enzyme to specific dna binding sites variants are designated by molecular weight the most common subunit is sigma 70
what is IPTG? what does it do?
IPTG is isopropylthiogalactoside and it works as an inducer of the lac operon (like allactose) where it is nonmetabolizable and is used experimentally
lacI encodes what? mutations cause what?
LacI encodes for the lac repressor protein and it has its own promotor (Pl) and mutations in lacI will result in the constitutive synthesis of the operon. Basically multiple operons are made even when the LacI is supposed to be repressing it
how can RNA be involved in transcription into DNA?
RNA can serve as a template for dna synthesis through reverse transcriptase it carries the genetic information that can be transcribed into DNA, and retroviruses like HIV or HPV convert the RNA in their genomes into DNA through reverse transcription (rna dependent rna polymerase) there is also a way by using telomerase to produce telomeres to protect the DNA at the ends of eukaryotes
in eukaryotes which polymerase synthesizes mRNA? what is needed for promotor region for eukaryotes?
RNA polymerase II eukaryotes require a TATA box located at the -25 5' end of transcription start site , but not sufficient promotor so additional elements are located between -40 and -110
describe the steps within RNA synthesis for binding and initiation how does this differ from DNA?
RNA polymerase binds to double-stranded DNA and searches for initiation sites called promoter motifs (TTGACA and TATAAT) where it will unwind a short stretch of double-helical DNA to produce a single-stranded DNA template RNA synthesis requires no primer
what effect will eating a death cap have on rna synthesis? what if it eat a lot of death caps?
RNA polymerase is unaffected by alpha-amanitin created by death caps, but in any dose, the alpha-amanitin will strongly inhibit polymerase ii and affect our ability to make mRNA in high doses, alpha amanitin will inhibit RNA polymerase III which will stop the creation of proteins since we cant transcribe 5S rRNA and tRNA
what needs to occur for RNAs to be functional? Before being transported from nucleus to cytoplasm what must occur to RNA
RNAs can be modified by nucleases, by excision of certain RNA segments, and/or by chemical modification of the RNA nucleotides RNA must be 5' capped, spliced, and polyadenylated before being exported from nucleus for translation in cytoplasm
what occurs during the splicing reaction? what role does U 6 and U4 play in this?
U2 binds adenine or the branching point and will add additional snrnps to form a phosphodiester bond at the adenine residue with G residue this forms a lariat that pulls the intron away and the exons together to create mature mRNA U6 is responsible for splicing reaction and U4 protects the cells from unnecessary splicing
what roles does U5 and U6 snRNPs provide? how about U4?
U5 binds the 5' splice site to the 3' splice site U6 catalyzes the splicing U4 protects the catalytic site from U6
how is the poly A tail added? where is it added
a poly A addition site is where endonuclease cleaves this site becomes an addition site for the poly A tail 10-30 nucleotides from the highly conserved sequence 5' (AAUAAA) 3' it is added upstream by 20-40 nucleotides is a sequence rich in G and U residues
what levels is transcription regulated? where is it closely regulated? how can transcription be repressed or activated?
at any step, and it is closely regulated at the first committed steps of transcription at the binding of the RNA polymerase to the promotor step it can be activated by cAMP receptor proteins but blocked by RNA synthesis at specific genes
the binding of transcriptional activators to enhancer elements causes ?
bending and looping of the DNA which allows the activation domain to interact with the basal transcription complex and produce an enhancement of transcription
what enzymes help with lactose metabolism in E.Coli? what are their function? why?
beta-galactosidase cleaves lactose to galactose and glucose and galactoside permease transports lactose into the cell this is because glucose is the preferred energy for bacteria but when lacking glucose they can use lactose for energy by maing more glucose
what are the steps of dna transcription?
binding of the rna polymerase to promoters initiation of transcription (make first nucleotides and initiation actors) elongation of transcription (elongation factors termination of transcription at specific sequences (termination factors (H)
the synthesis of RNA occurs in what stages?
binding, initiation, elongation, and termination
differentiate constitutive enzymes and induced enzymes what is enzyme repression?
constitutive enzymes are always present in constant amounts regardless of the metabolic state of the organism (will be turned off or on) induced enzymes are enzymes found in low amounts and can increase when their substrates are present this is when an enzyme is not synthesized because it is not needed
what happens to hNRNA?
heterogenous nuclear RNA is processed when the introns are spliced out and the mature RNA is now ready for protein translation
when lactose is high , glucose is low and cAMP is high what happens to the lac operon?
high transcription, since the repressor is unbound, the cAMP is inducing the lac operon and lactose is signaling for transcription as well
how is mRNA transcribed in eukaryotes? why is this box necessary in eukaryotes? IS it enough ?
mRNA in eukaryotes is synthesized by RNA polymerase II. The enzyme binds to a promotor region located at 5' of the transcription start site at -25 called the TATA box which is similar to -10 in prokaryotes The TATA box is necessary for polymerase II binding, but it is not strong enough and we will usually need additional elements between -40 and -110
what are the qualities of enhancer sequences? How does this differ from silencer regions?
may be long distances upstream or downstream of the start site must be on the same strand as the gene can be found within introns and can affect transcription regardless of it orientation silencer regions are the opposite and can silence a gene thousands of bases away, are found in introns, and can repress transcription while still on the same gene strand
What is the function of miRNA as opposed to siRNA?
miRNA = Translation inhibition siRNA = RNA cleavage
are cellular mRNAs degraded at the same rates? what does mRNA concentration govern? why is this important to the degradation of mRNA? what is the average halflife of mRNAs? which type of mRNA repeats have lower half-lifes?
no they degrade at different rates mRNA cellular concentration governs the level of gene expression degradation of mRNA ensures that mRNAs dont build up in the cell and direct the synthesis of unnecessary proteins average mRNA half life is 3 hours , and even less than this when the mRNA has an AUUUA repeat since the stability becomes lower, or when an mRNA has its poly A tail cleaved
do eukaryotes use the rho independent RNA synthesis? How do eukaryotes terminate transcription for mRNA?
no they do not use the rho independent rna synthesis hair loop, since tat is only used by prokaryotes to terminate mRNA transcription a conserved polyadenylate signal (AATAAA)is transcribed 10-15 nucelotides down for endonuclease to cleave the strand at site 10-15 bases downstream. A poly A tail is then added to cap it off
how does alternative transcript production occur in eukaryotes
pre-mRNA contains molecule signals for all the alternative processing pathways RNA binding proteins promote on a particular path and in this specific path complex transcripts can have more than one site where the poly A tails can form. This allows for the creation of different genes, and then the formation of a cap so it won't be degraded
differentiate eukaryote RNA polymerase from prokaryote RNA polymerase
prokaryotes will have RNA synthesized by the same RNA polymerase eukaryotes have 3 different RNA polymerases that differ in template specificity and RNA products
what are the bacterial repressors? how do they compare to each other?
repressors are proteins that block RNA polymerase binding or its movement along DNA operators bind sites on the DNA that bind repressors and are generally located near a promotor effectors are a small molecule or a protein that regulates repressor binding to DNA to alter transcription and will bind to the repressor and cause a conformational change
how is RNA synthesis termination signalled?
rho independent (no need for protein) terminators signals that has a self complementary region that forms a hairpin because they have a conserved string of 3 A residues that are transcribed into U residues near the 3' end of the hairpin this rapid folding will throw off the polymerase and stop the transcription like an anchor being thrown off a boat
the regulation of gene expression is achieved how?
specialized proteins and RNAs the proteins like ligand binding proteins with no other function but bind specific sequences in dna and rna. they respond to molecular signals that can be any kind of biological molecules and the rnas interact with other rnas or serve as cofactors
how is rna synthesized? Is it more complex in eukaryotes or prokaryotes
synthesized by RNA polymerases using DNA templates and ribonucleotide 5' triphosphates it is made in 5' to 3' direction and complementary to the template DNA strand in a highly regulated process that initiates the recruitment of transcription machinery to gene promotors. this process is much more complex in eukaryotes
What's another way we can change the proteins in translation? what is an enzyme that does this?
the RNA can edit itself to allow a different sequence to form. The different sequence will allow for a different protein to be made cytosine deaminase which is used to switch out the cytosine for a uridine
the cellular concentration of a protein is determined by what?
the delicate balance of at least seven processes each having several potential points of regulation the processes include synthesis of a primary RNA transcript, posttranscriptional modification of mRNA (introns and exons and promotors) degradation of mRNA, protein synthesis and posttranslational modifications of proteins, protein targeting and transport, and protein degradation
what happens when the rna polymerase lacks a separate proofreading 3' to 5' exonuclease active site? How about if it has an abnormal sigma 70 subunit?
the rna polymerase will lead to high error rate and an abnormal sigma 70 subunit will cause the dna polymerase to not be able to initiate the synthesis
what occurs in systemic lupus erythematosus? what role do snRNPs have in this?
the splicing is blocked by antibodies to snRNPs in patients with the autoimmune disease complexes of antibodies and nuclear proteins lead to arthritis, kidney damage, and skin abnormalities
if complementarity between miRNA and its target is nearly perfect what happens? if the complementarity is only partial what happens?
the target mRNA is cleaved, and if the complementarity is partial the complex will bind the translation of the target mRNA
what are housekeeping genes? What are examples of housekeeping genes? what is constitutive gene expression? what directly afects this?
these are genes for products that are required at all times and are expressed continuously enzymes of central metabolic pathways which have varying cellular concetrations of proteins expression of a gene at approximately constant levels the rna polymerase promotor interaction strongly influences the rate of transcription initiation
what are the qualities of transcription factors?
these are tissue specific and gene specific factors that turn on specific genes in specific tissues
how can mRNA be degraded faster? why is this important?
this can be done by removing the polyA tail or decapping it, another way is to endonucleatically cleave it all of which can lead to decay or exosome degradation of mRNA this is important so we dont have unneeded synthesis of proteins
what is an operon? what characteristics does its genes hold?
this is a gene cluster and promotor, plus additional sequences that function together in regulation operon genes in the same operon encode subunits of a larger protein complex or are involves in related processes that have coordinated regulation
what is a spliceosome? what are snRNAs? how are splice sites specified?
this is a large complex made up of multiple specialized RNP complexes called small nuclear ribonucleoproteins (snRNPs) and dozens of other proteins snRNAs are 100-200 nucleotide RNAs that make up snRNPs mRNA precursor splice sites are specified by sequences at the ends of the introns
what role does allolactose play? what happens when it binds lac repressor?
this is an isomer of lactose that works as an inducer of the lac operon when it binds the lac repressor it causes a conformational change and disassociation that results in lac operon gene expression
a gene can give rise to multiple products how? what is alternative splicing and how does it play a role with genes giving rise to multiple products?
through differential RNA processing which is where some mRNA transcripts are processed in more than one way to produce different mRNAs and thus different polypeptides alternative splicing is the process in which a particular exon may or may not be incorporated into the mature mRNA transcript which allows a small amount of genes provide myriads more proteins, since we are able to splice and create different codes based off the splicing that occurs
what are the transcription factors used by Polymerase III? which is the most important one?
transcription factor IIIA, IIIB, IIIC transcription factor IIIA is the most important and is the zinc finger
what are transcription factors? What are general transcription factors? WHy are they important
transcription factors are an array of proteins that work with RNA polymerase II to form the active transcription complex general transcription factors are the factors needed at every RNA polymerase II promotor they are important because they help the cell focus on what needs to be made since all cells have every gene in the body, it is to make sure we don't grow a toe in our armpit or a finger in our mouth
what are the four points dna is controlled in gene expression?
when DNA is being created into primary RNA transcript it has a transcriptional control there is a RNA processing control when primary RNA transcript becomes mRNA and after the mRNA leaves and is being used to translate protein there is a translation control. finally there is a protein activity control to turn the protein on or off
