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