Ch. 26

Front 1

transcription

Back 1

copying a gene/allele (DNA) as RNA

Front 2

another name for noncoding strand? is this one akin to the mRNA sequence?

Back 2

template strand, no

Front 3

is the coding strand akin to the mRNA sequence?

Back 3

yes

Front 4

in DNA, which strand (5' -> 3' or 3' -> 5') the template strand for DNA replication?

Back 4

both (can be flipped around and both can be replicated)

Front 5

in DNA, which strand (5' -> 3' or 3' -> 5') the template strand for DNA transcription? what is its formal name?

Back 5

3' -> 5', noncoding strand

Front 6

function of mRNA

Back 6

encode AA sequences of polypeptides

Front 7

function of tRNA

Back 7

match specific AAs to triplet codons in mRNA

Front 8

function of rRNA

Back 8

interact with tRNA during translation

Front 9

function of miRNA

Back 9

pos-transcriptionally regulate expression of genes by binding to mRNA sequences

Front 10

function of ribozymes- another name?

Back 10

found in ribosome to join AAs together and form protein chains; RNA splicing

catalytic RNA

Front 11

what does RNA polymerase require to function?

Back 11

- DNA template

-ATP, UTP, GTP, CTP

-Mg2+

Front 12

in which direction does RNA polymerase function? general steps?

Back 12

5' -> 3'

uses 3' hydroxyl (deprotonated) to attack alpha phosphorous of incoming nucleotide and releases PPi (pyrophosphate). reaction made more exergonic with breakdown of pyrophosphate into 2 inorganic phosphates using inorganic phosphatase.

Front 13

function of Mg2+ in transcription

Back 13

divalent cations in RNA polymerase:

1. stabilize transition state (electrostatic interactions)

2. shape active site to favor transition state

3. enhance electrophile activity of P

Front 14

how big is the transcription bubble?

Back 14

17 bp

Front 15

the recouperation after unwinding and rewinding of DNA in transcription requires the use of what enzyme?

Back 15

topoisomerase

Front 16

which end of the mRNA comes out of the transcription bubble first? why is this important?

Back 16

5' end, important because this is where 'post-transcriptional' modifications occur first (5' cap)

Front 17

what is the function of the NTP channel?

Back 17

funnels new NTPs to polymerase active site

Front 18

how is the correct RNA base pair chosen in transcribing DNA?

Back 18

base pairing (H bonding) and geometric shape (steric effects)

Front 19

the same sequence (with exception of U and T) occurs between which two strands of DNA and RNA?

Back 19

nontemplate (coding strand) of DNA and RNA transcript

Front 20

open reading frame

Back 20

part of DNA that is encoding genes (potential to be transcribed)

Front 21

if genes can be encoded on either of the 2 DNA strands, will the proteins produced be identical or different?

Back 21

different genes on either strand will produce different proteins

Front 22

how big is the RNA-DNA duplex?

Back 22

8 bp

Front 23

what happens to the DNA after the newly synthesized mRNA peels away?

Back 23

DNA duplex reforms

Front 24

is transcription (RNA polymerase) faster or slower than replication (DNA polymerase)?

Back 24

slower

Front 25

what is RNA polymerase made up of?

Back 25

5 core subunits: alpha2, beta, beta prime, omega

extra subunit: sigma

Front 26

function of 2 alpha subunits

Back 26

assembly and binding to UP elements

Front 27

function of beta subunit

Back 27

main catalytic subunit

Front 28

function of beta prime subunit

Back 28

DNA binding- routes DNA through channel

Front 29

function of omega subunit

Back 29

structural organization: holds everything together

Front 30

function of sigma subunit

Back 30

directs polymerase to promoter (brains), binds transiently

Front 31

how does RNA polymerase proofread its work?

Back 31

no proofreading ability! mistakes in RNA not critical because enough proteins are produced to sustain life

Front 32

why is sigma subunit said to bind transiently?

Back 32

it only directs polymerase to promoter then dissociates from core

Front 33

does RNA polymerase need a primer?

Back 33

no it has a promoter

Front 34

how big is RNA polymerase? where does it start and where does it end (numbering)?

Back 34

100 nucleotides

starts at -70 (5' end of new RNA, upstream) and ends at +30 (3' end of new RNA, downstream)

Front 35

promoter regions in E. coli; what sequence is at these regions? why is space between them important? what binds to them?

Back 35

-10 and -35

TATA box (Pribnow box)

if space between them changes, RNA polymerase can't bind properly

sigma

Front 36

where is the UP element located and what does it consist of? what binds to it? is it a constant part of the DNA?

Back 36

upstream of promoter (-40 to -60)

AT rich

alpha

not always present in all E. coli promoters

Front 37

how does sigma know where to land?

Back 37

-base specific interactions between major groove and polymerase's alpha helix

-associations with sugar phosphate backbone (stabilization)

-more interactions in minor groove to confer specificity

Front 38

how does the UP element affect sigma binding?

Back 38

it's a strong promoter that pulls the polymerase in so it lowers Kd. When it's not present, sigma doesn't bind as well --> RNA polymerase doesn't bind as tightly --> not as much protein produced

Front 39

how is sigma a point of regulation for gene expression?

Back 39

type of sigma varies based on the proteins/functions needed (s70, s45, etc)

Front 40

most common type of sigma and its function

Back 40

s70, housekeeping

Front 41

steps of transcription initiation

Back 41

1. polymerase binds to promoter (led by sigma) and forms closed complex

2. DNA is partially unwound near -10 and forms open complex

3. transcription initiated, complex changes conformation, drives Kd up, pushes sigma to dissociation

4. template strand moved through active site

*double-stranded primer not needed like in replication*

Front 42

what are the ways transcription initiation is regulated (before RNA is made)?

Back 42

-differences in promoter sequences (different sigmas needed)

-transcription factors (accessory proteins) binding near promoter (activators or repressors)

Front 43

how are biochemists able to capture photos of an elongation complex in action? what is the principle behind this maneuver?

Back 43

take away 3'OH, RNA polymerase is processive (can't let go of DNA until it's finished) but can't proceed without OH

Front 44

RNA polymerases cannot transcribe the same gene at once. true/false?

Back 44

false

Front 45

steps of p-independent termination

Back 45

1. self-complementary sequence of RNA forms hairpin structure

2. DNA sequence after hairpin contains AAA which is transcribed into uridylate at 3' end of RNA

Front 46

what do both terminations processes require?

Back 46

pausing of RNA polymerase on DNA transcript

Front 47

steps of p-dependent termination

Back 47

1. p protein binds to RNA at specific rut site and migrates 5' -> 3' until it catches up with polymerase (migrates using ATP hydrolysis because p is a helicase)

2. polymerase usually pauses at CA-rich region

might also contain a hairpin structure

Front 48

function of RNA polymerase I

Back 48

makes precursors of rRNA

Front 49

function of RNA polymerase II

Back 49

*the big one*

makes mRNA precursors

Front 50

function of RNA polymerase III

Back 50

makes precursors of rRNA, tRNA, etc

Front 51

largest subunit of RNA polymerase II and which structure it's analagous to in prokaryotic RNA polymerase? what is its distinguishing feature?

Back 51

RBP1

beta prime

long -YSPTSPS- tail imp for regulation (structure is more looped than like a helix or strand)

Front 52

why are so many more subunits needed in eukaryotic RNA polymerase II?

Back 52

packaging of DNA is far more complex

Front 53

what number is the TATA box at in eukaryotes? why can regulatory sequences be so far away from it?

Back 53

-30; DNA strand will fold over because the nucleosome

Front 54

4 steps of eukaryotic transcription

Back 54

1. assembly

2. initiation

3. elongation

4. termination

Front 55

what main event initiates transcription?

Back 55

phosphorylation of RNA pol II by TFIIH

Front 56

steps for eukaryotic transcription assembly

Back 56

1. TATA binding protein (TBP) binds to TATA box (-30)

2. TFIIF chaperones RNA pol II to correct TATA/Inr sequence

3. TFIIH completes closed complex by binding to RNA pol II and starts unwinding DNA to make open complex (helicase)

Front 57

steps for eukaryotic transcription initiation

Back 57

1. TFIIH phosphorylates RNA pol II's C-terminal domain (its OH-rich tail) causing conformation change (kinase)

2. TFIIE and TFIIH are released

Front 58

steps for eukaryotic transcription elongation

Back 58

TFIIF remains bound to RNA pol II and other factors bind to complex to enhance activity

Front 59

steps for eukaryotic transcription termination

Back 59

RNA pol II dephosphorylated

Front 60

when does post-transcriptional processing occur?

Back 60

during transcription

Front 61

do transcription and translation happen at the same time in prokaryotes? in eukaryotes? why or why not?

Back 61

yes in prokaryotes

not in eukaryotes because translation happens in cytosol not nucleus (which is why they need a 5' cap and why polycistronic mRNA in eukaryotes is not common)

Front 62

purpose of 5' cap

Back 62

protects mRNA from exonucleases

Front 63

what is the cap made of?

Back 63

7-methyl-guanosine

Front 64

when is the cap methylated? where is the methylation?

Back 64

after it has been added to mRNA at N7

Front 65

what four enzymes make up the cap-synthesizing complex? where is the complex situated?

Back 65

phosphohydrolase

guanylylytransferase

guanine-7-methyltransferase

2'-O-methyltransferase

C-terminal domain of RNA pol II

Front 66

what holds the cap to the polymerase?

Back 66

cap-binding complex

Front 67

steps of 5' capping

Back 67

1. start with 5' end of RNA (contains triphosphate) (NTP)

2. phosphohydrolase cleaves gamma phosphate off of RNA using general base catalysis (NDP)

3. guanylyltransferase deprotonates OH on beta phosphate (becomes nucleophile) to attack alpha phosphate on GTP (Gp-ppNp)

-PPi from GTP comes off

4. guanine-7-methyltransferase transfers Met from adoMet to Gp-ppNp (m7Gp-ppNp)

5. 2'-O-methytransferase transfers Met from adoMet to nucleotide P (m7Gp-ppmNp)

5. cap-synthesizing complex comes off and cap-binding complex tethers 5' cap to CTD of RNAPII

Front 68

what does the enzyme complex for the poly A tail contain?

Back 68

-endonuclease

-poly A polymerase

-proteins for sequence recognition, cleavage stimulation, regulation of poly A tail length

Front 69

irony of poly A tail function in bacterial mRNAs

Back 69

cause degradation instead of protecting from it like in eukaryotes

Front 70

steps of poly A tail addition

Back 70

1. enzyme complex binds to AAUAAA

2. mRNA trimmed back to ~20 nucleotides from AAUAAA sequence by endonuclease at 3' end

3. poly A polymerase adds 80-250 adenosine residues to trimmed transcript

Front 71

are introns or exons shorter?

Back 71

exons

Front 72

four intron groups and what they're spliced by

Back 72

group 1 (self-splicing)

group 2 (self-splicing)

spliceosomal (spliceosomes)

tRNA (protein-based enzymes)

Front 73

steps for group 1 introns

consensus sites?

Back 73

1. GTP, GDP, GMP's 3'OH is deprotonated (nucleophile) and attacks P at 5' end of splice site

2. newly joined exon-OH 3' attacks P at 3' end of splice site

3. intron degraded and exons fused

(transesterification)

no consensus sites

Front 74

steps for group 2 introns

consensus sites?

Back 74

1. 2' OH on intron adenosine is deprotonated (nucleophile) and attacks P at 5' end of splice site

2. lariat formed: contains 1 2',5' phosphodiester and 2 3',5' phosphodiester

3. newly joined exon-U-OH 3' attacks P at 3' end of splice site

4. intron degraded and exons fused

*2' OH because 3' OH is already involved in phosphodiester linkage

consensus sites 5' GU...AG 3'

Front 75

how many snRNPs does a spliceosome contain? one snRNP is associated with how many snRNAs?

Back 75

5, 1

Front 76

is ATP required for function of spliceosomes?

Back 76

no, just for assembly of spliceosome

Front 77

what makes up an inactive spliceosome? an active one?

Back 77

inactive: U1, U2, U5, U4/U6

active: U2, U5, U6

Front 78

steps for spliceosomal introns

consensus sites?

Back 78

1. U1 snRNA binds to 5' end of exon and U2 snRNA binds to sequence (includes A) nearer to 3' end

2. U4, U5, U6 bind and form inactive spliceosome

3. U1 and U4 dissociate and active spliceosome formed

4. A (on exon) in complementary U2 snRNA sequence is puckered out to form a good nucleophile

5. A attacks 5' end of intron and forms lariat

6. 3' OH on 5' end of intron attacks P on 3' end of intron

7. intron released as lariat and exons fused

*happens at CTD of RNA pol II since spliceosome is tethered at CTD*

Front 79

how is specificity conferred in spliceosomal introns? major groove interactions?

Back 79

no major groove since RNA is single-stranded; base pairing

Front 80

what transcribes rRNA? are rRNA molecules methylated?

Back 80

RNA pol I; yes

Front 81

is the transcription of rRNA or tRNA more complex?

Back 81

tRNA- lots of modification

rRNA is transcribed into 1 or 2 long genes then spliced