Nucleic Acids Set 6: Rna Processing Etc.

Front 1

What is one obvious difference wrt to prok euk transcription?

Back 1

Prok transcription and translation occur simultaneoously, and Euk TransC and TransL occur in diffferent cellular compartments

Front 2

How is cDNA made from Euk cells? What is the different b/w cDNA and regular DNA in Euks?

Back 2

cDNA is made using Reverse transcriptase on the mRNA molecule (doesn't happen in cells normally, usually in vitro

-cDNA is DNA complementary to the mRNA strand, so it doesn't have introns (which are present in the Euk genome)

Front 3

How could you figure out where introns are in DNA?

Back 3

use RT on the mRNA, and compare it to the genome

Front 4

HOw many sub unites does the RNA polymerase apoprotein have?

Back 4

5 (2 alphas, B', B, and omega)

Front 5

What does RNA polymerase do?

Back 5

It separates the DNA to form a transcription bubble (exposing 15-17 NTs) , and then begins RNA synthesis from 5'--3'

Front 6

How many RNA polymerases are in Euks? Which one do we need to know?

Back 6

3, We need to know RNA Pol II,

Front 7

What type of RNA does RNA pol II produce?

Back 7

snRNA, and mRNA

Front 8

Is the DNA overwound or under wound in front of the RNA polymerase?

Back 8

overwound

Front 9

Is the DNA overwound or Underwound behind the RNA polymerase?

Back 9

Underwound.

Front 10

What is the transcription start site known as? What is the numbering sequence upstream of the transcription start site?

Back 10

+1 site, it goes -1,-2,-3 (3'-->5') - No 0 position.

Front 11

What area is 5' of the +1 site, what important sequence does it contain?

Back 11

the 5' UTR, it contains a ribosome binding site (called the shine dalgarno sequence in Proks)

Front 12

Do Proks have TATA boxes at their promoters?

Back 12

NO, Euks have TATA boxes, Proks don't

Front 13

what are the sites of the promoters

Back 13

-10 and -35 sites

Front 14

What is an UP element?

Back 14

40-60 base pairs upstream of the +1, these are in proks, the prok RNA poly binds to these, and up elements are often in housekeeping genes that are constitutively expressed.

Front 15

What does the Prok RNA polymerase apoprotein (B, B', 2Alphas, and omega) need to become a holoenzyme? How does this change the RNA polymerase?

Back 15

It requires a sigma factor, which makes the RNA poly a holoenzyme. This holoenzyme can now recognize the -10 and -35 promoter sites specifically. W/o the Sigma factor, the DNA poly can bind to the DNA randomly, and not as tightly.

Front 16

Does it usually require ATP to unwind dsDNA into ssDNA?

Back 16

yes, this is usually performed by helicase in Euks

Front 17

How does is RNA polymerase able to bind to DNA? does this require ATP?

Back 17

Does not require ATP. It is thermodynamically favourable for RNA poly's sigma factor to bind to the promoter region.

Front 18

How does RNA poly move along the DNA?

Back 18

Sigma factor breaks off, and the DNA goes back to DS, as sigma is only specific to the promoter. NTPs are added to the growing RNA chain. A pyrophosphate group falls off, and this is what provides the free energy for the formation of (m)RNA

Front 19

WHat are the 2 types of termination in proks?

Back 19

Rho dependant and rho indépendant

Front 20

How does Rho dependent termination work? Where does Rho bind, on what molecule, how does it it stop mRNA synth, and does it require E?

Back 20

Rho binds to the Rho utilization sequence (RUT) on the growing mRNA strand.Once it binds to the Rut, it chases the RNA polyermase, and this requires ATP.

Front 21

How does Rho independent termination work?

Back 21

a hairpin structure forms in the growing mRNA strand due to the formation of a palindromic sequence. following this hairpin is a U-rich region, which allows the mRNA to dissociate from the RNA polymerase.

Front 22

How does the promoter regulate transcription?

Back 22

the stronger the promoter (closer to consensus sequence), the more transcription occurs. (not taking into account regulatory proteins, but we don't need to know.

Front 23

what is the difference between euk and prok DNA in terms of structure

Back 23

Euk DNA is composed of chromatin, DNA wrapped in histones. Proks don't have histones, they work with bare DNA

Front 24

What is the difference between Proks and Euks in terms of RNA processing

Back 24

Euks have introns in their mRNA that must be spliced out before it leaves the nucleus. Euk mRNA also gets fitted with a 5' guanine cap and a Poly A tail

Front 25

What type of extragenic transcriptional control occurs in euks but not proks

Back 25

Euks have enhancer and silencer sequences in extragenic regions Their genes are controlled by promoters as well. Proks just have promoters and Up elements (don't need to know about operator regions)

Front 26

how are histones removed from Euk DNA? Is this ATp dependent? WHat enzyme catalyzes this reaction?

Back 26

Requires ATP. A lysine residue on the histone protein reacts with Acetyl-Coa, producing CoA-SH and an Acetylated histone protein. This reaction is catalyzed by histone acetyltransferase.

Front 27

Which enzyme removes the acetyl group off of histones, and puts the histone back on DNA?

Back 27

Histone DeAcetylase

Front 28

which cofactor is required from both histone acetyltransferase and histone deacetylase?

Back 28

Chromatin remodelling complex

Front 29

What portion of RNA pol II is significant for co transcriptional processing in Euks, what does it do?

Back 29

The C terminal Domain (CTD) is phosphorylated, and it binds to, and brings RNA modifying complexes close to the growing RNA molecule.

Front 30

Where is the promoter in Euks? what's it called?

Back 30

the TATA box, located at the -30 site

Front 31

Compare the consensus sequence of the prok promoter to the Euk promoter

Back 31

Prok promoter = TATAAT

EuK promter = TATAAA

Front 32

what are some additional promoter elements the Euks have the proks don't?

Back 32

CAAT box - upstream of TATAAA -basal element

- GC Box- similar to CAAT box

-Response elements - similar to an operator- a molecule like a lipid may bind a protein, which will then bind a response element to regulate transcription

-Enhancers and silencers-interact strongly with promoter to regulate transcription

Front 33

WHat are transcription factors?

Back 33

Proteins that interact with Euk RNA pol II (TF II specifically ) for transcription. Some are specific for specific genes, and some are general for all genes.

Front 34

What are all of the transcription factors needed for RNA synthesis (not including function)

Back 34

TF II D (A), B,E,F,H

Front 35

What happens if we are missing one of the TF II transcription factors in a test tube?

Back 35

There will be no RNA synthesis.

Front 36

What is another name for TBP (tata box- Binding protein)- what does it do?

Back 36

TFII A or TFII D (same thing as TBP) - binds to tata box (i think)

Front 37

what does TFIIF do?

Back 37

it is a proccessivity factor, it makes sure the entire transcript gets produced.

Front 38

What does TFIIH do?

Back 38

It has helicase activity, it uses ATP to unwind the DNA so RNA poly can access it. If also phosphorlates the CTD (C terminal Domain) of RNA pol II, so it can interact with RNA processing proteins.

Front 39

What two activities to TFIIH have?

Back 39

Helicase and Kinase (as it can phosphorylate the CTD of RNA pol II)

Front 40

Which TF remains constant bound to RNA pol II, even after the RNA transcript is made?

Back 40

TFIIF

Front 41

What are the 3 ways the nascent mRNA transcript gets modified after synthesis by RNA pol II?

Back 41

addition of a 5' cap, splicing of introns, and 3' poly A tail addition

Front 42

What Nucleotide makes up the 5' cap? what is the full name

Back 42

7-methyl guanosine

Front 43

how is the 7-methyl guanosine linked to the 5' most nucleotide of the mRNA molecule

Back 43

a 5'-5' triphosphate linkage

Front 44

How many enzymes make the 5' cap?

Back 44

3

Front 45

What enzyme binds the 5' cap to the C terminus of the RNA Pol II?

Back 45

The CBC (Cap Binding complex)

Front 46

What is needed to direct where the 3' tail is added?

Back 46

a sequence at the 3' end of the mRNA molecule that signals a cleavage so a polyadenyl tail can be added.

Front 47

What is the sequence called that signals the cleavage for the poly A tail?

Back 47

the poly adenylation signal

Front 48

What adds the poly A tail to the nascent mRNA molecule? what is unique about this enzyme?

Back 48

Polyadenyl Polymerase- doesn't require a template because it just adds A's

Front 49

What is splicing?

Back 49

the process in which exons are linked together, and introns are cut out

Front 50

Why must splicing be very accurate?

Back 50

because if it is not, there is a high likelihood that a frameshift mutation will occur.

Front 51

What is alternative/differential splicing?

Back 51

It is when multiple proteins can be made from the same gene- there is a different combination of exons added to make the final mRNA molecule.

Front 52

What is a spliceosome composed of?

Back 52

a group of proteins composed Protein and snRNA (small nuclear RNA)

-snRNA is needed to ensure the sequences for splicing line up properly.

Front 53

How do Spliceosomes know where to cut? (what is the sequence they look for)

Back 53

They look for a 5' GU and AG 3' . THe ends of the intron are the the Gu and the AG

Front 54

What shape does the spliced out intron leave the mRNA molecule as?

Back 54

a lariat.

Front 55

What is the order of nucleophiles that is responsible for removing the intron

Back 55

the hydroxyl group of an Adenine residue within the intron attacks the 5' Guanine residue (GU), which then becomes a nucleophile, which attacks the 3' guanine molecule (AG), and this sequence is removes as a lariat structure.

Front 56

What type of bond joins the loop of the lariat structure?

Back 56

5', 2' phosphodiester bond.

Front 57

What do both Group I and Group II introns have in common?

Back 57

They are both Self Splicing. 0The catalyst is the RNA itself.

Front 58

What are group I & 2 introns?

Back 58

Ribozymes- RNA enzymes that do not require ATP

Front 59

What is the difference between group I and II introns?

Back 59

Group I introns leave as a linear molecule, and group II introns leave as a 2',5' phosphodiester bound lariat structure.

Front 60

Where is the Ribozyme in the ribosome, and what does it do?

Back 60

It is in the active site of the peptidyltransferase and ispart of the 23 SrRNA in the large subunit

Front 61

What is RNA editing?

Back 61

In some tissues, there is different protein expression from the same genes transcribed. What can happen, is an enzyme will "mutate" (change the base) somewhere in the pre-mRNA sequence, which will introduce an early stop codon, producing a smaller protein. A base can also be inserted or deleted, but this is not noticeable within the DNA, it only happens to the pre-mRNA .

Front 62

What does RNAi stand for? and siRNA?

Back 62

RNA interference . Small Interfering RNA

Front 63

What protein recognizes dsRNA in the cell, and what does it do

Back 63

DICER recognizes dsRNA and cleaves it into 20-22 nt fragments

Front 64

what enzyme recognizes these 20-22 nt fragments, and how does it recognize them

Back 64

RISC recognizes the 20-22 nT fragments cut by dicer based on size. RISC forms a complex with the small RNA molecule to make an RNA endonuclease which is specific to RNA molecules that have this sequence.

Front 65

How does the Cell use the RISC protein to stop the production of a certain protein.

Back 65

the nucleus produces microRNAs, small dsRNA molecules. these molecules are cleaved by dicer to produce 20-22 NT RNA fragments that bind to risc to prevent protein synthesis from RNA molecules that contain this microRNA sequence.

Front 66

What type of RNA molecules can be made from RNA editing.

Back 66

mRNA, rRNA, tRNA, microRNA,