Biochem Ii - Lecture 9

Front 1

READING: To what does RNA Poly add a nucleoside triphosphate (ATP, CTP, UTP and GTP) and release PP(i)?

Back 1

A growing RNA chain

Front 2

T/F: Eukaryotic cells (not in this particular lecture) contain 4 or 5 RNAPs that each synthesize a different class of RNA.

Back 2

True

Front 3

Name the literal subunit composition of the RNAP holoenzyme and what is unique about sigma.

Back 3

Alpha 2, Beta, Beta', omega, and sigma;

The sigma-factor dissociates from the core enzyme and carries out the actual polymerization process

Front 4

Which strand of DNA is transcribed?

Back 4

The antisense strand

Front 5

RNAP binds to its initiation sites through base sequences known as _ that are recognized by the corresponding σ factor.

Back 5

Promoters

Front 6

What do numbers indicate in labeling the promoter region of DNA?

Back 6

+1 is the FIRST nucleotide that is transcribed to RNA, there is no 0, and everything in the 5' direction (opposite of the way RNAP is moving) has a negative number

Front 7

T/F: RNAP binds to the -20 to +20 region tightly and protects it from degradation by DNases.

Back 7

True

Front 8

T/F: The rates at which genes are transcribed vary directly with the rate at which their promoters form stable initiation complexes with the holoenzyme.

Back 8

True

Front 9

Promoter mutations that increase or decrease the rate at which the associated gene is transcribed are known as _ _ and _ _.

Back 9

Up mutations and down mutations

Front 10

What technique showed scientists that the RNAP holoenzyme mainly makes its contacts with DNA at the -10 and -35 regions, on the same side of DNA?

Back 10

DMS footprinting

Front 11

On what strand of DNA is transcription performed?

Back 11

The antisense strand

Front 12

How long is the promoter of transcription and where is it located relative to the transcription site?

Back 12

~40bp, and it is located 5' of the transcription site

Front 13

Which promoter site is at the -10 position? What are the general base sequence here?

Back 13

The Prinbow Box (TATAAT)

Front 14

What sequence of base pairs is at the -35 promoter site?

Back 14

TTGACA

Front 15

What promoter consensus sequence is located at about -40 to -60, what base pairs is it rich in and why, and what does it do?

Back 15

The Upstream Promoter (UP) element, rich in A-T base pairs because they promote unwinding due to less stability than G-C, and it binds to the alpha subunit of RNAP

Front 16

Where is the transcription bubble complex located on DNA, and why does the formation of this open complex explain why promoter efficiency tends to decrease with the number of G-C pairs in the -10 region?

Back 16

Starting at about -10 region and 14bp long to just past the initiation site (+1); the open complex shows that A-T pairs are preferred for proper unwinding and thus transcription because they are less stable than G-C pairs

Front 17

Core enzyme does not specifically bind promoter DNA except when…

Back 17

It has a UP element

Front 18

Why does core enzyme bind to duplex DNA so much more tightly than holoenzyme RNAP, i.e. RNAP that contains the sigma subunit?

Back 18

Because when in it's holoenzyme form, apparently the sigma subunit allows for looser binding and thus quicker movement along the DNA duplex for recognition of the sigma promoter and thus quicker transcription

Front 19

Once transcription has begun and the sigma subunit has been removed from the holoenzyme, what does the core enzyme do?

Back 19

Binds to and stabilizes the enzyme-RNA-DNA complex.

Front 20

T/F: Unlike DNA replication, the initiation of the formation of an RNA chain simply involves the coupling of two nucleoside triphosphates and thus does not require a primer.

Back 20

True

Front 21

Thus, what is the structure of RNA in prokaryotes in terms of phosphates?

Back 21

On the 5' terminal end there are THREE phosphates from the initial chain initiation reaction and throughout the rest of the molecule there is only one alpha-phosphate in the chain.

Front 22

What is the process called when RNAP releases newly synthesized RNA after only ~10nt have been polymerized?

Back 22

Abortive initiation

Front 23

Why does abortive initiation occur?

Back 23

It occurs because RNAP keeps its grip on the promoter region (on the sense/nontemplate strand) while pulling the antisense/coding strand through its active site creating the "scrunching" phenomenon: the tension from this is sometimes released by just releasing the newly synthesized, short strand of RNA prematurely

Front 24

T/F: The sigma subunit dissociates and forms the elongation complex catalyzing the polymerization of the new RNA strand, and then in a cyclic manner reattaches to the core enyme forming the holoenzyme and performing RNA-chain initiation at a promoter region once again.

Back 24

True

Front 25

T/F: Nucleic acid scrunching often precedes the transition from the initiation complex to the elongation complex.

Back 25

True

Front 26

What is the complex called in which DNA has not yet entered the main channel to form a transcription bubble?

Back 26

A closed complex

Front 27

How does Rifamycin stop RNA chain initiation altogether?

Back 27

It binds to the RNAP holoenzyme on the Beta subunits, not allowing the RNAP to dissociate from the promoter and thus not allowing RNA chain initiation.

Front 28

T/F: The RNA chain only transiently forms a short length of RNA-DNA hybrid duplex, as is indicated by the observation that transcription leaves the template duplex intact and yields single-stranded RNA. Thus, the unpaired transcription bubble of the DNA in the open initiation complex apparently travels along the DNA with the RNAP.

Back 28

True

Front 29

What is the correct model for the winding of RNAP and DNA during transcription?

Back 29

RNAP moves in a straight line while positive supercoiling occurs ahead of the transcription bubble and negative supercoiling occurs behind it so as to keep the linking number unchanged.

Front 30

T/F: Fun Fact - The synthesis of RNAs that are needed in large quantities, e.g. ribosomal RNAs, is initiated as often as is sterically possible, about once per second.

Back 30

True

Front 31

The binding of what antibiotic tightly binds to duplex DNA inhibiting both DNA replication and transcription, severely distorting the DNA helix to almost A-conformation and blocking elongation? What specific interactions is it associated with?

Back 31

Actinomycin - it has interactions with G-C base pairs

Front 32

About half of the transcription terminators in E.coli are occur without assistance. What are the 2 names for them and what are their 2 main characteristics?

Back 32

Intrinsic or spontaneous terminators; (1) they contain a tract of 7-10 consecutive A-T's with the A's on the template strand, and (2) just upstream of this they contain a G and C rich, palindromic segment.

Front 33

T/F: The stability of a terminator's G+C-rich hairpin and the weak base-pairing of its oligo(U) tail to template DNA are important factors in ensuring proper chain termination.

Back 33

True

Front 34

What protein enhances the termination efficiency of spontaneously terminating transcripts as well as inducing the termination of non spontaneously terminating transcripts?

Back 34

Rho factor

Front 35

What is the main thing that the Rho factor does, and how does it get the energy to do this?

Back 35

Unwinds RNA-DNA and RNA-RNA, which it does by utilizing NTPase activity and hydrolyzing NTPs into their NDPs + P(i).

Front 36

How does Rho attach to its substrate, ultimately releasing it?

Back 36

It attaches to the NEWLY FORMED RNA a the C-rich "rut" site and moves along the nascent RNA until it reaches the paused RNAP, releasing RNA

Front 37

LECTURE: T/F: RNA is synthesized by an RNA polymerase that catalyzes the 5'->3' polymerization of nucleoside triphosphates (NTPs: ATP, GTP, CTP & UTP) into an RNA polymer.

Back 37

True

Front 38

A primary transcript that was made de novo by RNAP at a specific recognition sequences thus chemically starts with what and ends with what?

Back 38

Starts with pppA or pppG and ends with the 3'OH

Front 39

DNA is locally denatured at the transcription start site to form an _ _ even before the first few nucleotides are added.

Back 39

Open complex

Front 40

Again, what are the 4 phases of transcription?

Back 40

Template binding, RNA chain initiation, chain elongation and chain termination

Front 41

T/F: The orientation of transcription units on DNA is not fixed; on some genes, the top strand is the template strand and on others it's the bottom, although all genes are transcribed in the 5'-->3' direction.

Back 41

True

Front 42

T/F: The synthesis of RNAs that are needed (such as rRNA_ can be initiated as often as is sterically possible (about once per second).

Back 42

True

Front 43

What are the "untranscribed spacers" between large sections of RNA in a micrograph of transcription?

Back 43

Promoters

Front 44

In transcription, about the -40 to +20 regions (promoter) will be covered by RNAP and thus protected from digestion by DNases. What does the -10 region consensus sequence of the promoter actually promote?

Back 44

Melting open of the DNA

Front 45

What is arguably the most important subunit of RNAP and what does it do?

Back 45

The sigma subunit, it recognizes the promoter and is involved in transcription initiation

Front 46

What are the factors that improve the fidelity of RNAP by cleaving stalled transcription elongation complexes about 2-10nts from the 3' end called?

Back 46

GreA and GreB

Front 47

T/F: Although there are many mutations in promoter regions across prokaryotic species, in general, the closer the promoter sequence is to the consensus, the more efficient the level of transcription.

Back 47

True

Front 48

What are the actual consensus sequences at the -35 and -10 regions of prokaryotes?

Back 48

-35: TTGACA; -10: TATAAT

Front 49

Why aren't weak promoters necessarily a bad thing?

Back 49

Because they allow gene expression to be up and down-regulated.

Front 50

In the experiment of enzymatic footprinting of RNAP on DNA, what was trying to be shown?

Back 50

Where RNAP binds to DNA and thus where the promoter regions are

Front 51

RNAP scans the DNA and binds at promoter sequences forming a... Then, the sigma subunit induces the formation of an _ _ within the promoter at approximately the -10

Back 51

Closed complex

Front 52

After transcription is actually initiated, what are the two possible outcomes and what happens in the latter?

Back 52

It can be aborted (about half the time) or it can enter the elongation phase, in which RNAP releases the sigma subunit and take on the nusA subunit for recognition of termination signals

Front 53

Where does the energy for forming the open complex in transcription initiation come from?

Back 53

The release of negative supercoiling

Front 54

Briefly describe Rho-independent termination.

Back 54

The new RNA forms a G-C rich hairpin, leaving only a dA-rU helix which is very unstable and leads to the RNA release

Front 55

The signal for transcription termination resides in the...

Back 55

Nascent RNA

Front 56

How does Rho-dependent termination basically occur?

Back 56

It occurs when Rho binds the nascent RNA (preferring C-rich, unstable regions) and moves 5'-->3', catching up with paused RNAP, and unwinding the RNA-DNA helix using ATP hydrolysis, finally releasing the transcript

Front 57

How do Rifamycin and Actinomycin work to stop transcription?

Back 57

Rifamycin binds the RNAP Beta subunit and block transcription initiation; Actinomycin binds the DNA template and thus blocks elongation

Front 58

What happens to the expression of the sigma subunit when bacteria are exposed to heat?

Back 58

A heat shock sigma factor (sigma^32) is expressed instead which recognizes the same promoters and other ones the the normal sigma one does, so that enzymes that help heated proteins fold can be expressed

Front 59

Describe how phages like SPO1 use an exchange of sigma factors to control their own DNA transcription.

Back 59

SPO1 uses host sigma factor to transcribe early genes, the product of early genes to get a new sigma factor that binds to middle genes, and so on for late genes.

Front 60

Contrastingly, what does T7 phage do?

Back 60

It makes its own RNAP in its early genes which then transcribes later genes (which moves about 6 times faster than E. coli RNAP!)

Front 61

T/F: Phage-made polymerases have a common 3D structure that mimics E. Coli DNAPoly I.

Back 61

True

Front 62

When T7 RNAP goes from the initiation to the elongation phase, it changes conformation greatly. What are these main three changes?

Back 62

(1) the promoter binding site is destroyed

(2) A channel for the 7bp heteroduplex is created in the active site

(3) an RNA exit tunnel is created