biochem unit 7
describe how riboswitches can terminate transcription, block translation and interfere with splicing. are riboswitches an example of cis or trans regulation
"cis regulation" because mRNA regulates its own translation, a part of the molecule regulates another part aptamer binds proteins creating conformational changes to disrupt the 3 things
recount briefly three mechanisms which control the activity of enzymes in the cell
(1) allosteric proteins (2) proteolytic cleavage (3) covalent modification
discuss two distinguishing features of the termination signal in the mRNA in rho independent termination of transcription in prokaryotes
(1) region that produces @ 3' end of RNA transcriptwith self-complementary sequences, permitting hairpins (2) highly conserved string of three A residues in template that's transcribed into U residues near 3' end of hairpin so when polymerase enters this termination site, it pauses and formation of (1) hairpin disrupts A=U (2) base pairs in RNA-DNA segment and causes dissociation between RNA and polymerase
point out features at exon-intron junction that provides signals for correct splicing. what are the consequences of imprecise splicing
(5')GU....AG(3') -U1 snRNP is complementary to GU(5') -U2 is paired to intron @ position encompassing A residue that becomes nucleophile during splicing reaction
define the number system which is used to identify regions such as the -10 and -35 regions
+ numbers refer to DNA base pairs regions after the transcription start site - numbers are given to DNA bp before transcription start site (promoters are usually -70 to +30)
what are transcription factors?
-only for eukaryotes -other proteins that polymerase needs to form active transcription complex in eukaryotes
discuss the key steps in the addition of a poly(A) tail. is the tail encoded in the gene? name the enzymes involved in making the poly(A) tail. does this enzyme add residues in a template directed manner like polymerase?
-steps: the transcript is extended beyond site where poly(A) tail is added but the extra site is cleaved by endonuclease, cleavage generates free 3'-OH group where polyadenylate polymerase adds A -the tail is not encoded in the gene -enzyme: polyadenylate polymerase -it does not add in a template directed manner
list four important features that distinguish regulation of gene expression in eukaryotes from prokaryotes
1) access to eukaryotic promoters is restricted by structure of chromatin and activation of transcription is associated with many changes in chromatin structure 2) positive mechanisms predominate in all systems in eukaryotes though it has both + and - 3) eukaryotic cells have larger, more complex multimeric regulatory proteins 4) transcription in eukaryotic nucleus is different in space and time from translation in cytoplasm
discuss the formation of a primary transcript and its processing to an mRNA. draw it
1) transcription and 5' capping 2) completion of primary transcript 3) cleavage, polyadenylation (3' poly-A tail), splicing
discuss the quaternary structure of RNA polymerase
5 core subunits: alpha, beta, beta, omega the two Beta subunits form pincers a 6th subunit, sigma directs enzyme to specific binding sites on DNA theta subunit makes it so RNA pol isn't so strong at binding
draw a diagram of the lac operon. label each of the following: operon, promoter site, operator sites, structural genes. DRAW IT
5'-Pi, lacI, O3, P, O1, lacZ, O2.....lacY, lacA-3'
what percentage of the human genome is believed to encode protein? how much of our genome is currently believed to be transcribed into RNA? what is a "TUF"?
<2% of genome encodes proteins if repetive sequences are excluded, at least 40% of the remaining genomic DNA is transcribed into RNA TUF=transcript of unknown function
compare DNA and RNA with respect to pentose ring and nitrogen bases
DNA: deoxyribose, ACTG RNA: ribose, ACUG
does splicing take place at the level of DNA or RNA?
RNA
is RNA usually double or single stranded? does single stranded RNA have any double stranded regions?
RNA is usually single stranded but can have double stranded region known as hairpin loops (usually palindromic)
distinguish between RNA polymerase and DNA polymerase I in terms of substrates, whether a primer is needed, the direction of synthesis, the nucleophilic attack in bond formation, the role of pyrophosphate and pyrophosphatase in overall reaction, the requirement for a template
RNA polymerase uses NTP, no primer, 5-3, 3' hydroxyl acts as nucleophile and attacks phosphate of incoming NTP, pyrophosphate is used to drive the rxn forward and pyrophosphatase cleaves pyrophosphate to 2 Pi, template is needed note DNA is same except dNTP, primer is needed
clearly distinguish between RNA processing and RNA editing
RNA processing: adding poly(A) tail, splicing RNA editing: adding or deleting bases in coding regions of primary transcripts
using 26-8 (p1065) describe the sequences typically found at a eukaryotic promoter. how do these sequences compare to those found at prokaryotic promoter?
TATA box: TATAAA initiator sequence (Inr): YYAN(T or A)YY where N=any nucleotide and Y=any pyrimidine TATA box=major assembly point for the proteins of preinitiation complexes of Pol II (general transcription factors) DNA is unwound at INR both eukaryotic and prokayotic sequences at promoters hae lots of A-T pairs--it's easier to unwind
how do individual snRNAs locate the consensus sequences that flank introns?
U1 snRNP is complementary to (5')GU U2 snRNP is paired to intron at position encompassing the A residue
point out the location and identity of the nucleotide that will make the initial attack on the 3' end of the exon.
adenylate 2' OH residue near 3' end of intron will attack phosphoryl group of G @ 5' splice site
define specificity factor
alter specificity of RNA oplymerase for a given promoter
distinguish between poly A site choice and alternative splicing (DRAW IT). discuss the role of each of these mechanisms in generating protein diversity from a single gene.
alternative splicing: cutting different # of introns out of the sequence makes different mRNA products poly A site choice: mRNA has multiple sites where it can add poly A tail, choosing one over the other results in multiple mRNA products
discuss the role of basal transcription factors, transcription activators, chromatin modification and remodeling proteins, coactivators
basal transcription factors: general transcription factors form a pre-initiation complex at TATA box (help bring polymerase to right site) transcriptional activators: bind to enhancers or upstream activator sequences and helps facilitate transcription activity and rate chromatin modification and remodeling protein: opens up chromatin by nucleosome positioning, covalently bonding nucleosomes, histone charges coactivators: MEDIATOR, facilitates communication between activators and complex @ promoters
define operator
binding sites that repressors bind to
what is the function of the sigma subunit?
binds transiently to core and directs core to correct binding site on DNA, which is important for initiation of transcription
distinguish between constitutive and regulated gene expression
constitutive: unvarying expression of a gene regulated: cellular levels of gene products rise and fall
is DNA usually double or single stranded
double stranded
define positive regulation
enhancing the activity of RNA polymerase at promoter
distinguish between exons and introns
exons are expressed. introns are spliced out.
what is meant by RNA world? what evidence is used in support of hypothesis?
first, simple molecules became nucleotides when electrical discharges gave energy, nucleotides became RNA->self-catalyzing RNA was selected for->proteins were especially made because proteins were better at catalysis and DNA as more stable evidence: RNA catalysts exist, wide range of rxns for which RNA catalysts have been developed, RNAs that are vestiges of RNA world, RNA catalyst is responsible for synthesis of proteins, RNA can self-catalyze
how does cAMP play a role in positive regulation of lac operon
glucose->no CAMP-> no CRP-> no Lac transcription when glucose is low, you have high cAMP no glucose->CAMP->CRP->Lac transcription
distinguish between the following DNA-binding motifs: helix-turn-helix, zinc finger, homeodomain and leucine zippers
helix-turn-helix: recognition helix binds to major groove, 2 alpha helixes, one of which is specific for DNA zinc finger: Zn stabilizes the fold but still weak homeodomain: 60 AA, transcriptional regulator especially during eukaryotic development leucine zipper: amphiphatic alpha helix with series of hydrophobic, AA on one side, have Leu lined up side by side (Leu=+ which is good for DNA)
distinguish heterochromatin and euchromatin
heterochromatin: very condensed, cannot transcribe euchromatin: not condensed, can transcribe
describe how the lac operon can be negatively regulated. what protein is involved? name the gene that encodes this protein. what molecule acts as the inducer?
in absence of lactose, lacI encodes lac repressor protein. Repressor binds to O1 and O2/O3. The intervening DNA (either P or lacZ) are then repressed and cannot be transcribed. when there is lactose, allolactose acts as the inducer, which causes conformational change in repressor that causes dissociation->allows lactose expression
use fig.26-6 (pg 1061) to discuss the events in transcription initiation
initiation: RNA polymerase core binds to DNA promoter and sigma subunit guides them there, transcription bubble forms, transcription is initiated elongation: base pairs continue to be added on. sigma dissociates and is reaplced by NUSA. termination: transcription finishes. NUSA dissociates, RNA polymerase is recycled
discuss the structural features of active chromatin
it's open so you can transcribe it
name each of the structural genes and describe the function of each
lacI: encodes Lac repressor lacA: encodes B-galactosidase (breaks down lactose) lacZ: encodes permease (allows lactose to enter) lacY: encodes transacetylase (gets rid of harmful products)
write an equation representing the hydrolysis of lactose to its component monosaccharides. what is the name of the enzyme which catalyzes this reaction.
lactose->glucose+galactose enzyme=B-galactosidase
what is the function of the poly(A) tail?
located @ 3' end, most eukaryotic mRNA has string of 80-250 A residues in eukaryotes, tail protects mRNA from enzymatic destruction
note what RNA tertiary structure looks like
look at picture
discuss the synthesis and processing of miRNAs. what is RNAi?
miRNA: silences genes precursor mRNA is cut by Dicer to become stRNA; complement is removed by helicase; single strand is complementary to strand you want to silence RNAi: RNA interference
describe and list two functions of the 5' cap added during the processing of eukaryotic mRNA
modified nucleotide (usually guanosine) linked by triphosphate bridge 1) protects mRNA from ribonucleases 2) participates in binding of the mRNA to the ribosome to initate transcription
distinguish between monocistronic and polycistronic mRNA. is eukaryotic mRNA and prokaryotic DNA monocistronic or polycistronic?
monocistronic DNA: only carries code for one polypeptide polycistronic DNA: carries code for multiple eukaryotic=monocisronic, prokarotic=polycistronic
do all eukaryotic genes contain inrons? do most?
most contain introns
does the same strand always serve as the template strand?
no
does RNA pol have 3-5 exonuclease activity?
no because RNA polymerase is only transient anyway; it's going to get degraded eventually
what is the "lariat structure"
noose-liike structure formed by a nucleophilic attack; formed by 2',5' phosphodiester bond
what is the relationship between the "strength" of a promoter (i.e. the frequency at which transcription is initiated at a particular promoter) and the degree of match to a consensus sequence?
one bp being off in a conenseus sequence can decrease rate of binding by several orders of magnitude
describe the key features of "operon" are operons found in prokaryotes or eukaryotes? do they encode monocistronic or polycistronic mRNA?
operons have activator binding site, promoter, operator/repressor binding site found in both monocistronic DNA
define activator
protein that enhances RNA polymerase promoter interaction by binding to enhancer
define repressor
protein that impedes access of RNA polymerase to promoter; bind to operators
how do proteins interact at specific DNA sequences? what type of interaction is most often involved? within the DNA double helix, where does this interaction occur?
proteins interact at major groove with H-bond and hydrophobic interactions
describe the function of rRNA, mRNA and tRNA
rRNA: component of ribosome (carries out synthesis of proteins mRNA: carries genetic info from genes to ribosome tRNA: translates info from mRNA into specific sequence of amino acids
is transcription usually regulated at the level of initiation, elongation or termination?
regulation can happen at any time but is mostly at initiation
what is an enhancer
regulatory sequence generally bound transcription activators
define negative regulation
repressor protein blocking transcription
define catabolite repression
restricts expression of genes required for catabolism of lactose in the presence of glucose
what is a "consensus sequence"
short sequences that are commonly found
what is a spliceosome? what is an snRNP?
spliceosome: large protein complex made up of snRNPs, specialized RNA-protein complexes snRNP: contains 1 of a class of eukaryotic RNA, 100-200 nucleotides called snRNA
what is meant by template and non-template strands?
template: serves as template for RNA synthesis non-template strand: complementary to DNA strand, will be the exact same as RNA you're coding
what is meant by chromatin remodeling?
transcription-associated structural changes of chromatin including 1) positioning of nucleosomes 2) presence of histone variants 3) covalent modification of nucleosomes
what is the significance of the -10 and -35 regions?
two consensus sequences are found at these two places that are found in lots of promoters
how does cAMP receptor protein (CRP) play a role in positive regulation of lac operon
when glucose is absent, CRP binds to site near the promoter and stimulates RNA transcription, acts as a POSITIVE REGULATORY ELEMENT