240 test 3

Describe the 3 phases of transcription

- Initiation: getting the process started (bringing the necessary proteins to the appropriate locations on the DNA, adding the first nucleotides into place -Elongation: polymerizing (adding onto the chain) -Termination: stop polymerization and detach proteins involved from DNA

Know the consensus sequences targeted for splicing

-5' splice site GU -Branchpoint A -3' splice sight AG

Mediator complex

Helps to recruit RNAPII complex the first time and for subsequent rounds of transcription

Transcriptional regulation

How cells control whether or not gene is expressed, and at what levels. Specific DNA sequences (upstream of transcriptional start site) bind specific proteins. These proteins mediate expression of the gene.

Transfection

Introduction of foreign DNA into eukaryotic cells

mRNA processing

RNA transcripts is spliced and modified to produce mRNA which moves from nucleus to cytoplasm

Ribosomal RNA

RNAs that are important for the structure and function of ribosomes

Be able to diagram/explain how & why the trp operon is regulated at the levels of: -Transcription initiation -Transcription elongation/termination

Level 1: When tryptophan is present in the cells it will bind to a repressor which will bind to the DNA ahead of the promoter, inhibiting transcription of trp. (Either on or off). But what happens if we want a more fine-tuned regulation of Tryptophan production? -We go to level 2 regulation which is called TRP ATTENUATION. Level 2: Attenuation Reduction of Transcription: If the ribosome is fast (because there are lots of tRNAs loaded with tryptophan loaded in the cell) the ribosome will cover codon 2, and codon 3 & 4 will bind and form a hairpin. This TERMINATES transcription...and by default the translation of the protein will be left unfinished. Transcription Keeps Going: If the ribosome is slow (because there are few tRNAs loaded with tryptophan in the cell), the ribosome will stall before codon 2, which will allow 2 & 3 to bind and form a hairpin. This inhibits 3 & 4 from terminating transcription, but it will allow translation to continue...and as a result you will also get translation of the protein.

Polyadenylation site

Site that is cut and a poly A is added, half way between AAUAAA and U-rich sequences

fusion protein

The protein product of a gene created by the fusion of two distinct genes or portions of genes.

DNA binding domain

The site on a DNA-binding protein that directly interacts with specific DNA sequences.

RNA processing

caps, poly A tails and splicing

Transesterification

chemical reaction used by splicing. Occurs at A

activation domain

co-regulatory proteins bind to transcription factors

Promoter deletions

delete parts of promoter sequence, put into cells, and see if reporter enzyme is still made. Find what part has the biggest impact.

Sigma Factor

directs core enzyme to bind specifically to certain gene promoters

Inducer

disables repressor

Diagram the organization of the sequence elements at the 3' end of pre-mRNA that are involved in termination

do it

Co-activator

do not bind to DNA directly, but help distal DNA-bound TFs to interact with basal TFs at the promoter

Linker scanning mutagenesis

don't delete promoter sequences replace them with other DNA sequences having no regulatory function

R2D2

double stranded RNA binding protein

DICER

double-stranded RNA trimming enzyme

Downstream

down from where transcription starts, indicated with + number

Draw/describe the process of how introns are removed

ectron pair on the branch point (A) 2'-Oh attacks 5' phosphate on splice sight, (the G), breaking that phosphate bond to ribose and forming bond to 2'OH on creating Lariat structure • 3' OH of exon 1 will attack hte 5' phosphate on exon 2

explain how rRNA and tRNA are encoded in genome

encoded next to each under control of operon, helps with effectiveness

Argonaute

enzyme that cuts mRNA

core enzyme

enzyme that stick to DNA initially, able to polymerize RNA from some types of Dna, no discrimination when to start, 5 subunits

TATA box

equivalent of -10 promoter in prokaryotes, farther away in eukaryotes

Peptidyl transferase

forms peptide bonds between adjacent amino acids

Trp attenuator

forms secondary structures (stem loops)

Open reading frame

from start codon to stop codon

constitutive gene expression

gene is always expressed (mRNA transcription always occurs)

inducible genes

gene is normally inactive, but it can be activated

Describe the major events of transcriptional termination in eukaryotes, including the ability to distinguish between the 2 currents models for how it occurs

general steps § Cleavage of pre-mRNA at poly(A) site § Addition of poly(A) tail § Termination of transcription and removal of RNA polymerase ○ Factors involved in cleavage and poly(A)addition are carried to the poly(A) site by the RNAPII complex ○ Two models § Antitermination model: antitermination factors keep RNAPII, these factors replaced upon reaching poly(A) sit, promoting termination Torpedo model: Exonuclease grabs onto piece of RNA that is cut after poly-A site. The exonuclease runs into RNAPII, kicking it off.

RNA pol II basal transcription complex

group of proteins that binds to promoter regions and brings RNAPII to begin transcription

Anti-codon

group of three bases on a tRNA molecule that are complementary to an mRNA codon

Charged tRNA

has amino acid attached to it

Upstream

head of where initiation begins (+1 indicated first nucleotide that is transcribed), will be indicated with - number

Enhancer (aka, upstream activator sequence or UAS)

increase efficiency of gene expression by recruiting of mediator complex

shRNA

like a miRNA, but produced artificially

Regulatory promoter

located further upstream (5') than core promoter, site where transcriptional activator proteins bind, activators bind to regulatory promoters and assist TRIID to recruit rest of basal transcriptional complex

Predict/draw the RNA sequence from a labeled dsDNA template sequence

look up (unit 19)

Terminator

means that transcription stops here (RNA sequences that kick off RNA polymerase)

RNA Interference

mechanism used to control gene expression after a cell has committed to making a mRNA by cleaving specific mRNA before they are translated or blocking accessibility to ribosomes

miRNA

microRNA, long RNA which bends back on itself to form a hairpin. Precursor of siRNA, encode multiple siRNA

RNA splicing

modification of the nascent pre-messenger RNA (pre-mRNA) transcript in which introns are removed and exons are joined prior to translation.

tRNA and mRNA have.....end mrna has

monophosphate, triphosphate

Epigenetics

patterns in gene expression that are controlled by heritable but potentially reversible changes in Chromatin structure

Oligo(dT) colun

purifies mRNA from all other RNAs based upon the poly(A) tail

Explain how rRNA and tRNA are encoded in the bacterial genome and why they are organized in that fashion

rRNAs surround tRNAs in the genome. They need to be in the cell in equal amounts.

core promoter

region upstream of a gene where the basal transcription complex begins to assemble

Promoter

regions upstream of genes and are the site of transcription initiation. Transcription factors and RNA polymerase bind here to initiate transcription

Codon

sequences in gene that codes for amino acid

siRNA

short interfering RNA, final RNA product after processing miRNA/sHRNA. Starts double stranded stranded, and remaining strand is guide strand

Define UTA

untranslated region, non-coding sequences. 3' where siRNA can bind to stop transcription, u Rich

Primer extension

used to determine start site of RNA transcription

Guide strand

used to find specific mRNAs for silencing

Reporter Assay

used to measure the activity of various enhancer or promoter elements. Luciferase takes a substrate and converts it to visible light. Place gene for luciferase behind promoter

self-splicing

uses protein complexes (no snRNAs) to assist in the process of removing introns, catalytic activity in RNA itself.

Know which enzymes/proteins are necessary for mRNA processing and nuclear export

• 5' capping RNA 5' triphosphatase/guanylytransferase (capping enzyme) • Poly-A tail: Poly(A) polymerase • Splicing: spliceosome • Nuclear export ○ Cap binding complex Poly(A) binding protein

Describe the purpose of the Yeast Two-hybrid Assay

• A way to rapidly screen for unknown proteins that interact with a particular protein of interest • Based upon production of chimeric proteins

Describe the main attributes of transcriptional activators

• Bind to DNA directly at regulatory promoters and enhancers (not to core promoters, that is for basal transcription complex) • Contain activation domains to assist in recruiting RNA pol II • Recruit RNA polymerase II inderectly through mediator complex • Necessary for efficient transcription • Many types, play big role

Holoenzyme

RNA polymerase enzyme with the sigma factors.

Explain what components are necessary for transcription to begin and end (both DNA/gene components and polymerase components

-Begin: RNA polymerase, promoter, sigma factor, core enzyme -End:Rho protein binds at rut site in mrna and runs into RNA polymerase, signals polymerase to stop transcribing .Rho-independent termination of transcription: a hairpin forms, as it exits the the polymerase it signals it to let the polymerase go

the major players in the lac operon

-Beta-galactosidase: converts lactose to galactose and gucose -Permease: Brings lactose into cell Transacetylase: transfers acetyl CoA to lactose -Repressor: keeps lac operon turned off -Inducer: turn lac operon on when necessary -Promoter: RNA polymerase binds to promoter to transcribe if no repressor blocking the operator -Operator: repressor binds to promoter/operater in order to block access to promoter

Be able to differentiate between the 3 main control regions for transcription: core promoters, regulatory promoters, and enhancers (function, general location)

-Core Promoter: Located in the direct vicinity of the transcriptional start point Site where the RNA polymerase and associated transcription factors bind to initiate transcription Very similar for all genes transcribed by RNA pol II, because same basal transcription complex used for all genes transcribed by RNA pol II -Regulatory Promoter Located further upstream (5') than the core promoter Site where transcriptional activator proteins bind (activator proteins vary from gene to gene) Activators bind to regulatory promoters and assist TFIID to recruit the rest of the basal transcriptional complex -Enhancers Their role is to increase the efficiency of gene activation Can be far away from the promoter Can be upstream or downstream of the promoter, and even in an intron Still function when removed and reinserted in the opposite orientation

Capping

-Modified guanosine attached to 5' end -Needed for mRNA to exit nucleus and bind ribosome -CH2 binds o phosphate

summary of eukaryotic transcription

-Specific activator proteins bind to specific regulatory promoters and/or enhancers when transcription of specific genes under their control is needed by the cell -Mediator complex gets signal from activators (see above) to bring in RNAPII and the rest of the basal factors to the core promoter: basal complex is now assembled -Another activator bound to a regulatory promoter makes contact with the basal transcription complex, thus signaling RNAPII to transition from initiation phase to elongation phase -Some genes have enhancers that allow for very high levels of transcription by strongly recruiting the mediator complex repeatedly to keep bring in more RNAPII

Describe the relative activity of transcription of the lac operon based upon any combination of lactose or glucose

-Very low: lactose is absent and glucose is abundant -Low: low glucose and lactose, starvation mode, cell is desperate to find any energy source, repressor still on, cAMP is bound, some transcription when repressor breathes off -Middle: high glucose and lactose, cells prefer to use just glucose if it is present. -High: low glucose and high lactose, using as energy source, CAP and CAMP turbocharge.

Describe the general function and structure of rRNA

-structural component of ribosomes (essential for proper structure) -Participate in the chemical reaction of forming peptide bonds in a growing chain of amino acids

Diagram/explain how and why the trp is regulated at the level of initiation, elongation/termination

-transcription initiation: on or off, no fine tuning. Tryptophan binds to the repressor when there is plenty present, allowing it to block gene expression -Elongation/termination: fine tuning of expression, dependent on the speed of ribosomes. if ribosome moves slow, 2:3 and transcription can continue. If ribosome is fast, it covers 2, 3:4 stem loop forms, and transcription aborts in the leader sequence.

levels of operon activity based on levels of glucose and lactose

-very low if lactose absent and glucose abundant -Low if both are absent, cell desperate to find energy source. As repressor breathes on and off, get a little expression because cAMP is bound (it recruits RNAP) -middle if lactose and glucose are present. cell prefers to just use glucose instead of cleaving lactose to glucose -high lactose is present, no glucose. cAMP intensifies transcription

Describe and differentiate between the 2 main methods used to make DNA more accessible to transcription. Also, how nucleosomes are changed by these methods

. Chromatin remodeling—moving histones to new locations on the DNA molecule. --Displace from molecule, or translocate to new position on same molecule Nucleosome modification—acetylation or methylation of basic amino acids in the histone tails (nucleosome does not move). Acetylation add NH3 to NHCC, giving neutral charge. Methylation NH3+ to NH3+CH3 no impact on charge, hinders polymerase from transcribing --If you remove the + charge, the DNA wound around the histone becomes more loose

Describe the 3 steps involved in eukaryotic mRNA processing

. Addition of 5' caps: Guanosine nucleotide added to 5' end of mRNA via triphosphatase/guanylytransferase. 2. Addition of 3' poly-A tails: PAP=poly-A polymerase adds a string of adenines onto the 3' end of the mRNA. 5' cap and 3' poly(A) tail functions: Stability of mRNA Nuclear export of mRNA to cytoplasm Promote translation 3. Splicing out of introns: Spliceosomes splice out the introns from the mRNA. Splicing function: Greater protein diversity from a single gene Tissue-specific protein production --Since different tissues will splice the same mRNA different ways, different cell types can use the same gene for different purposes

Explain why and how the presence of glucose regulates the lac operon

.If glucose is present, the cell will not want to waste energy by breaking Lactose down into glucose. Lactose is broken down into glucose IF the cell does not already have glucose to use as energy.

Explain/list the main differences in Eukaryotic vs. Prokaryotic gene expression (focus on transcription)

1. # of RNA polymerases used: 3 in euk. 1 in prok. 2. complexity of RNA polymerase: more subunits in eukaryotes 3. Prokaryotic RNA polymerase binds to DNA directly and stays there; Euk. doesn't and must be called in 4. Euk. have a nucleus and must export mRNAs to cytoplasm. Prok. transcribe and translate at the same time 5. Euk. have nucleosomes and chromatin; prok don't 6. mRNA processing only in euk

Identify 5 types of alternative splicing

1. An optional exon can be skipped or included 2. Only one exon in an array of optional exons may be included in mature mRNA 3. An intron may be retained or excluded 4. An exon might have alternative splice sites at its 3' end 5. An exon might have alternative splice sites at its 5' end

Differentiate between methods to map out DNA sequences involved in transcription initiation

1. DNA affinity chromatography for purification of DNA-binding proteins a. Identify DNA sequences that bind proteins at all b. Identify specific proteins that bind to DNA sequences of DNA 2. Deletion of various promoter regions a. Find which regions change function 3. Linker Scanning mutagenesis can be used to determine regulatory regions a. Upstream segments of a gene are replaces with a linker sequence of equal size to determine if gene can be transcribed 4. Chip assay: have proteins bind to DNA in chips 5. Reporter assays Chromatography and CHIP look at proteins, others at DNA

List the categories of transcripts produced by the 3 Eukaryotic RNA polymerases

1. RNA polymerase I (14 subunits): transcribe large rRNAs 2. RNA polymerase II (12 subunits): transcribe mRNAs and some snRNAs 3. RNA polymerase III (17 subunits): transcribe tRNAs, small rRNAs, other non-coding RNAs

Identify/distinguish between the 2 categories of bacterial enzymes used to degrade mRNAs.

1. Ribonucleases: cleave phosphodiester bonds using hydrolytic cleavage, endonuclease and exonucleases. 2. Polynucleotide phosphorylases break phosphodiester bonds by adding in a second phosphate group, work in 3' to 5 direction. Need PAPI enzyme to get past 3' hairpin structures.

Explain how mature/functional tRNA is trimmed from larger transcripts

1. Trim from 3' end, end with CCA and 3' OH 2. Trim from 5' end, end with GGG and 5' monophosphate 3. Modify bases to become unusual bases a. RNASES can't recognize them 4. Add and amino acid to the sugar at the 3' OH end to charge the molecule

Describe methodology of yeast 2 hybrid assay

1. clone the cDNA for your protein of interest into a plasmid vector so that it is adjacent to a DNA sequence encoding a DNA binding domain (DBD). This is a fusion protein and is plasmid 1 2. Transcriptional activating domain bound to one member of cDNA library (plasmid 2) 3. Plasmid 3 encodes for protein necessary for histidine biosynthesis (yeast needs it to survive) and promoter region. This promoter is activated if proteins produced from interaction between plasmids 1 & 2 interact with each other Introduce plasmids to cells. Extract DNA from colonies that grow, determine cDNA sequence that allows colonies to grow. Identify the gene and protein produced, verify with 2nd experiment

Operon

2 or more contiguous ORFs under the control of a single operator

Transcription factor

:a molecule (usually a protein) that regulates whether or not transcription will take place or not take place at specific genes

spliceosome

A large complex made up of proteins and RNA molecules that splices RNA by interacting with the ends of an RNA intron, releasing the intron and joining the two adjacent exons.

Repressor

A protein that suppresses the transcription of a gene.

Acetylation

Acetylation: add an acetyl group, removing the positive, repelling them from each other

Polyadentylation

Adding a polyA tail to the 3' end

Describe the purpose of RNA interference and how we can manipulate the process for therapeutic benefit, and the mechanisms

Because sometimes the cells have buyer's remorse and want to silence genes after they already transcribe them. RNA interference: a way to regulate gene expression by cleaving specific mRNAs before they are translated or blocking accessibility to ribosomes MicroRNA (miRNA): this is a long RNA which bends back on itself to form a hairpin. It is the precursor molecule to make siRNAs. These occur naturally in the cell and often encode multiple siRNAs. Short hairpin RNA (shRNA): this is just like a miRNA (also a precursor), but it is produced artificially in the laboratory in order to manipulate gene expression. Short interfering RNA (siRNA): this is the final RNA product after processing miRNA. Starts out double-stranded, but then one strand is removed and the remaining strand is the guide strand. Guide strand is complementary and anti-parallel to the target mRNA. This is the molecule used to find specific mRNAs for silencing. DICER: Double-stranded RNA trimming enzyme. Cleaves miRNA to form siRNAs. R2D2: Double-stranded RNA binding protein. Helps the dicer bind to miRNA. HELICASE: kicks off passenger strand GUIDE STRAND: functional strand, determines specificity (which mRNA molecule to silence?). Complementary and parallel to the target mRNA genes that are meant to be silenced. ARGONAUTE: Enzyme that actually cuts the target mRNA. (remember it carries the sword -> warrior)!

polynucleotide phosphorylase

Breaks phosphodiester bonds by adding in a second phosphate groups, works in 3' to 5' direction *exonuclease Need second enzyme to get past 3' hairpin structures common in mRNAs, which block PNPase

Operator

Component of a promoter that gives additional control over whether or not gene is actively transcribed

cDNA

DNA produced from mRNA

Monocistronic

DNA sequence that encodes a single ORF (1 protein)

Heterochromatin

More dense DNA, less accessible for RNA transcription (mostly repeated sequences, few genes, low activity)

Explain how unusual bases get incorporated into tRNA and why they get incorporated

Normal base is either modified or removed Present in loop, protect from degredation

Distinguish between the mechanisms used to splice out nuclear introns vs. self splicing introns

Nuclear introns use spliceosomes and a branchpoint. Self splicing : Do not use a spliceosome, but cleave themselves out. Connect one end to the other. AN exogenous G nucleotide initiates the first reaction in self splicing introns, no branchpoint or secondary structure. Juxtaposition of intron ends is mediated by secondary structure of pre-mRNA. Base-pairing assists in finding right place to splice. • External proteins are required to splice out group I and II introns effectively, but snRNAs and spliceosome are not involved

Learn the elements of a gene that control the starting and stopping of transcription and be able to explain the meaning of the + and - notions relative to the promoter

Promoters: upstream of transcriptional start point, site where RNA polymerase and transcription factors bind to start transcription (has TATA box, TTGACA, and startpoint). Terminators kick off RNA polymerase, ending transcription. RHO dependent termination (Rho protein binds at rut site of transcript, runs into RNAP, stopping it) RHO-independent forms a hairpin, ending transcription. + indicates downstream from start, - upstream

Explain the advantages of alternative splicing

Protein diverse proteins from a limited amount of DNA sequence

RISC

RNA induced splicing complex, includes DICER, R2D2, Guide strand of siRNA

alternative splicing

Splicing of introns in a pre-mRNA that occurs in different ways, leading to different mRNAs that code for different proteins or protein isoforms. Increases the diversity of proteins.

Describe the structure of the 3'polyA tail

String of adenine

Be able to draw/label the names and locations of the core promoter area for eukaryotic RNA pol II (only actual DNA sequence to know is TATA; slide 4)

TATA box:At ~ -25-30bp upstream of start site, where the DNA is opened. Initiator (InR): the sequence surrounding and including the start site for transcription. Transcription starts at +1 Downstream promoter element (DPE): At around +28 to +32

Polycistronic

a single gene (single promoter) that encodes multiple ORF (multiple proteins)

Euchromatin

The less condensed form of eukaryotic chromatin that is available for transcription.

Branchpoint

The middle A nucleotide where splicing occurs

Differentiate between the mechanism of RNA transcription and DNA replication

Transcription does not need primer, need more (unit 19)

Repressible genes

a gene is normally active, but it can be de-activated

Reporter Gene

a gene that researchers attach to a regulatory sequence of another gene of interest (Luciferase)

Methylation

adding methyl group, hinders polymerase from transcribing DNA

trp attenuation

allows the bacterial cells to be in an intermediate state of tryptophan production.

Activator

bind to regulatory promoters and enhancers to provide signal to move from transcriptional initiation to elongation

general transcription factors

bind to specific sequences in DNA, some of these then recruit RNA Pol II to the appropriate sites

Transfer RNA

bring amino acids to ribosomes for protein production, decipher genetic code by matching anticodons in tRNA sequence to codons in mRNA sequence.

polyadenylation signal

site that is initially recognized in termination, and required for cleavage and poly(A) addition. AAUAAA

snRNA

small nuclear RNA

snRNP

small nuclear ribonucleoproteins; recognize the splice sites in the pre-mRNA; located in the nucleus and are composed of proteins and RNA

Initiator sequence

surround and includes the start site for transcription

Describe the general function and structure of tRNA

tRNA: decipher the genetic code by linking together an RNA anti-codon (in tRNA) to the RNA codon in mRNA, deliver appropriate amino acids to proper place in ribosome for subsequent addition to a peptide chain E site: empty tRNA site P site holdes peptidyl tRNA A site holds Aminoacyl tRNA (like acceptor)

In Vivo

takes place in a living organism

In Vitro

test tube transcription and translation: use cell lysates to make proteins without using living cell

RNA polymerase

transcribes DNA to RNA

Differentiate between 3 main control regions for transcription

• Core promoters: located in direct vicinity of transcriptional start point, site where RNA polymerase and associated transcription factors bind to initiate transcription, very similar for all genes transcribed by RNA pol II, because same basal transcription complex used for all genes transcribed by RNA pol II • Regulatory promoters: located further upstream (5') than core promoter, site where transcriptional activator proteins bind, activators bind to regulatory promoters and assist TRIID to recruit rest of basal transcriptional complex Enhancers: can be far away from promoter, can be upstream or downstream, still function in different orientation, gene specific function

List 4 common DNA-binding motifs and be able to explain why there are a limited number of such motifs

• DNA structure not diverse, so common structural motifs 1. Helix-turn-Helix a. 2 parallel helices with a turn before the 3rd. 3rd helix inserts into the major groove, helix 1 and 2 interact with other proteins 2. Zinc fingers a. Peptide chain held together by zinc 3. Leucine zippers Helix-loop-helix

Describe how primer extension assay works to map the initiation of transcription

• Gene specific primer binds to DPE add reverse transcriptase, build to end of the RNA. Perform DNA sequencing on CDNA, • OR run through gel to find out how many bp there are, walk back from DPE to find +1 site

Describe major features of how the complex proceeds from the initiation phase to the elongation phase of transcription

• RNA PolYY c-terminal domain (CTD) • Every Eukaryotic RNA pol II contains multiple copies of a repeated 7 amino acid sequence at C-terminus of its largest subunit • CTD must be phosphorylated by TFIIH to trigger elongation 1. Nascent RNA transcript begins to push against TFIIB 2. RNA chain growth leads to TFIIB dissociation from polymerase 3. The 5' end of the ten residue transcript is no longer part of the DNA:RNA hybrid and begins t thread into the exit tunnel 4. Transcription complex escapes from promoter 5. TFIIB, TFIID, TFIIA remain bound to the promoter to facilitate subsequent rounds of transcription

Describe necessary events for effective eukaryotic transcription initiation

• Specific activator proteins bind to specific regulatory promoters and/or enhancers when transcription of specific genes under their control is needed by the cell • Mediator complex gets signal from activators (see above) to bring in RNAPII and the rest of the basal factors to the core promoter: basal complex is now assembled • Another activator bound to a regulatory promoter makes contact with the basal transcription complex, thus signaling RNAPII to transition from initiation phase to elongation phase • Some genes have enhancers that allow for very high levels of transcription by strongly recruiting the mediator complex repeatedly to keep bring in more RNAPII

What are the general transcription factors:

• TFIID: ○ TBP binds to TATA boxes TAFS bind to lnr and DPE • Binds DNA (the TATA box) • TRIIA: stabilized TFIID (prevents from falling off) • TFIIB: binds complex and recruits RNA Pol II • TFIIF: prevents nonspecific DNA binding. Binds RNAPII and is involved in recruiting RNAPII in the preinitiation complex • TFIIE: binds promoter near transcription initation site. May help to stabilize transcription bubble in the open complex • TFIIH: functions in transcription and DNA repair. It has kinase and helicase activities and is essential for open complex formation • DAB stays at core promoters, FEH moves with RNAP

Describe functions of each of RNAP II transcription factors in the basal complex and the order in which they assemble to form the basal complex

• TFIID: ○ TBP binds to TATA boxes TAFS bind to lnr and DPE ○ TBP+TAF=FIID • Binds DNA (the TATA box) • TFIIA: stabilized TFIID (prevents from falling off) • TFIIB: binds complex and recruits RNA Pol II • TFIIF-Pol2: comes with poly II to help it bind to TFIIB • TFIIE: binds TFIIB and recruits TFIIH to complex • TFIIH has helicase activity and kinase activity-opens template and phosphorylates RNA Pol II • DAB stay at promoter, FEH go with RNA

Explain the idea and general premise of the histone code

• There may be a code for histones, allowing us to predict if a gene will be active or inactive • Acetylated tails may be transcriptionally active, phosphorylated may prepare DNA for division ○ Sometimes acetylation can mean turned off, methylation on

Describe methods to study transcription and transcriptional regulation and their applications.

• Use RT-PCR to find presence of gene, time consuming • Clone the regulatory regions that control gene expression • Artificially fuse one genes promoter to another gene's coding sequence and still get production • reporter assay ○ Luciferase under the control of the promoter, if there is green, the promoter is on

Describe the structure of the 5' cap

○ Guanosine nucleotide added to via 5' to 5' linkage ○ 5' carbon of cap attached to 5' carbon of first nucleotide ○ Some 2' methyl groups at 5' end of mature mRNAs as well Triphosphate present

Describe components of the spliceosome and the purpose of the internal RNA molecules

○ U1 sits on top of 5'end of intron (bind to site) ○ U2 finds branchpoint A (bind to site) ○ U6: pairs with U2 and U5, mediates OH group on branchpoint A attackig 5' end of intron. Helps 3' end of exon 1 find 5' end of exon 2 -Internal RNA molecules identify branchpoint and splice site • snRNPs: snRNAs + various proteins called U1 snRNP, U2 snRNP etc.