Molecular Bio Exam 1 (post practice exam)

which polymerase can identify and fix transcription errors

RNA polymerase 2

which serine must be phosphorylated for elongation

serine 2 (S5 can decrease)

what is the purpose of phosphorylation of serines in the CTD

initiation of transcription

what is the amino acid sequence on the CTD of RNA polymerase 2

what is the function of TBP and where is it found

TATA box binding protein binds to the minor groove of TATA box and causes the TATA box to be bent into an 80 degree curve

TF2A

binds around TATA box w TF2D to form DA complex

TF2B

binds to DA complex forming DAB complex by bind to TBP through its C-term and to RNA polymerase 2 through N term

TF2D

binds to TATA and DNA along with TF2A to form DA complex which also contains TBP and TAF2s

TF2E

binds to DAPpollF complex along with TF2H forming DAB pollFEH which is required for promoter clearance

TF2F

helps RNA polymerase bind to a region from -34 to +17 thus forming DAPpollF complex

TF2H

binds to DABpollF complex along with TF2E forming DABpollFEH initiation complex which leads to phosphorylation of CTD domain and unwinding of DNA to form transcription buble

TF2S

stimulates transcription elongation by resolving transcription pauses

where is TATA box

-25

where is CAAT box

-80

where is GC box

-100

what type of supercoils does DNA gyrase help rewind

negative

what is the -10 region of prokaryotic bacteria rich in

A/T

what subunits of the eukaryotic RNA polymerases are involved in DNA binding when recognizing the promoter

NONE!!!! eukaryotic RNA polymerase is recruited by transcription factors that bind to the DNA

what subunits of the E. coli polymerase are involved in DNA binding when recognizing the promoter?

alpha (regulatory sequences), beta’ (-35 region), sigma (specific promoter regions)

what is the reason for minimal regulation in bacteriophade genomes>

its role is to get in and GET OUT

what is the first nucleotide incorporated in Bacteriophage promoters? Why?

G bc it is more thermodynamically stable due to the triple hydrogen bonds

what entity has many conserved nucleotides in their promoters

all different bacteriophages

why does a T7 bacteriophage gene have high affinity for RNA polymerase?

it is a structural gene (class 3)

what is the +1 nucleotide of T7

G

where is the TATA region in T7?

directly upstream of the +1 nucleotide

where is the TATA region in prokaryotic cells

~ -10

where is the TATA region in eukaryotic cells

~ -25

what recognized the TATA region in T7

specificity loop on RNA polymerase

what recognized the TATA box in prokaryotic cells

sigma factors

what recognizes the TATA box in eukaryotic cells

TBP on TF2D

how many polypeptides are in prokaryotic cells

5 (2a, 1b, 1b’, sigma factor)

how many polypeptides are in eukaryotic cells

10 + Rpb4/7

what terminates transcription of T7

hairpin that decreases the affinity of the RNA polymerase for the DNA

what terminates transcription in rho independent prokaryotic cells

hairpin like bacteriophage Rho

what terminates transcription on Rho dependent prokaryotic cells

rho factors binds at the rho binding site on the RNA, travels towards RNA polymerase, adn causes dissociation of the entire complex

what are probable amino acids that you would find on the surface of teh RNA polymerase protein?

what amino acid gets acetylated, methylated, and ubiquitinated

lysine

what enzymes adds acetyl groups

HATs

what enzyme rem

what does the addition of an acetyl group do

increased transcriptional activity

what enzymes methylates

HMTS

what does cytosine methylation do

represses gene activation

what is usually around the centromere of repressed genes

methyl

H3K9 methyation

transcriptional repression

H3K4 methylation

transcriptional activation

where does phosphorylation get the phosphate from

ATP

where does ubiquitination occur

H2A, H2B

which two histone modifications require ATP?

ubiquitination, phosphorylation

what causes the formation of heterochromatin

Xist

how does top 2 work

top will clamp down on the un-cut strand of DNA before the cut is made

what is the repeated sequence in telomeres

TTAGGG

to which stand does telomerase add nucleotides

template stand

what template does telomerase use to perform reverse transcription

RNA

to what strand does TER bind

leading

what synthesizes the lagging strand

TERT

what can you modify to switch heterochromatic to euchromatin

DNA and histone proteins

what is the first step in isolating protein

cell lysis (mechanically or chemically)

what step comes after cell lysis

centrifugation (to remove insoluable materials)

what comes after centtrifugation

differential precipitation with ammonium sulfate

what comes after differential precipitation

purification by chromatography

what comes after purification by chromatography

SDS (to add uniform neg chage)

what comes after SDS

gel electrophoresis

what comes after gel electrophoresis

transfer of protein to transfer membrane

what comes after the transfer of protein to transfer membrane

primary and secondary antibody binding to the protein

what comes after the primary and secondary antibody bind to the protein

incubation period with blotting substrate

what biomolecule gets lysed at very unfavorable conditions

RNA (to degrade RNAses)

what characteristics of RNA/DNA allow DNA to be separated from RNA using a CsCl density gradient?

RNA is denser than DNA, thus it will pellet in CsCl density gradient

which procedures for isolating uses ultracentrifuge to separate contents by size

CsCl

what isolation procedure uses the negative charge on DNA/RNA to attach to the positive silica

silica binding

what isolation procedure precipitates DNA and RNA in alcohol

alcohol precipitation

what happens to RNA at high pH

2’ OH deprotonates

what happens to RNA at low pH

purines are hydrolyzed off the ribose

why does protein purification require a different series of purification steps than RNA or DNA? How can you redily remove insoluble material and cell membranes?

protein purification requires cell lysis under very gentle condition to prevent denaturation and insoluble material/cell membranes need to be centrifugated.

how can you elute the protein of interest from column chromatography

by either increasing the concentration of a ligand or adding another ligand with higher affinity to the column.

what will bind in anion exchange chromatography

negatively charged proteins

what is the length of DNA

33.2A per 10.4bp

average amino acid molecular weight

110Da

pKa of -COOH

2-3

pKa of NH3+

8.5-9.5

σ⁷⁰

general principal sigma factor

σ⁵⁴

nitrogen regulation when deficient

σ³²

heat shock gene transcription