Lecture 19

Front 1

In the mismatch repair mechanism, what distinguishes the template and newly synthesized strand?

Back 1

Methylation (N6 of Adenine)

Does not change base pairing properties.

Front 2

Dam methylase

Back 2

Methylates the Adenine of the daughter strand.

Front 3

What is the window of opportunity in the mismatch repair mechanism?

Back 3

The period in which the template and daughter strand can be distinguished.

Before Dam Methylase methylates the daughter strands.

Front 4

MutL-MutS

Back 4

Recognizes mismatch
DNA is threaded through it till methylate adenine; knows the correct strand.

Front 5

MutH

Back 5

Cleaves the daughter strand (nonmethylated)

Front 6

In the mismatch repair mechanism, what DNA is degraded?

Back 6

Degrades all DNA from methyl group through the mismatch

Front 7

What are the major mechanism for DNA repair?

Back 7

1)Mismatch Repair
2) Base Excision Repair
3) Nucleotide Excision Repair
4) Direct Repair

Front 8

Which repair mechanism works best for deamination?

Back 8

Base Excision Repair

Front 9

DNA glycosylase

Back 9

Specific for particular each lesion
Recognizes damaged base and removes the base by cleaving the N-glycosyl bond.

Front 10

AP site

Back 10

Site at which no base is present but the rest of the backbone is.

Front 11

AP endonuclease

Back 11

Removes the sugar phosphate (base-less sugar)backbone at the AP site.

Front 12

In the base excision repair mechanism, what enzyme fills in the gap after removal of the base-less sugar?

Back 12

DNA polymerase I in the 5 to 3 direction.

Front 13

In the base excision repair, what is the last step?

Back 13

Nick is sealed by ligase.

Front 14

What is nucleotide excision repair used for?

Back 14

To remove large DNA lesion such as pyrimidine dimer (cyclobutane dimer caused by UV radiation)

Front 15

Xeroderma Pigmentosum

Back 15

Defects in the nucleotide excision repair mechanism (excinuclease protein specifically) prevents the removal of cyclobutane thymine dimer that form kinks

Front 16

Excinnuclease Complex

Back 16

Nucleotide excision repair mechanism:
1) Recognizes DNA lesion
2) Cleavage on either side of the lesion

Front 17

What are the final steps of the nucleotide excision repair after excinnuclease complex activity?

Back 17

Repair gap with DNA Pol I ( DNA pol e in humans) and the seal nick with DNA ligase.

Front 18

What's special about direct repair?

Back 18

Does not replace damaged DNA and repairs directly.

No turnover, so COSTLY!

Front 19

When is direct repair used?

Back 19

Alkylation of base changing base-pairing properties.

Front 20

What's difference between transcription and replication?

Back 20

During transcription, only particular genes are transcribed at one time. Machinery needs to identify these regions. Furthermore, only one strand is transcribed.

Front 21

What enzymes are needed to carry out transcription? What is the directionality?

Back 21

RNA Polymerase

Front 22

What are the differences between DNA and RNA polymerase?

Back 22

RNA polymerase does not need a primer and there is no exonuclease activity.

Front 23

What are the similarities between DNA and RNA polymerase?

Back 23

They both require DNA template, have 5' to 3' polymerase activity, form 5-3 phosphodiester bonds and require NTP as substrates.

Front 24

What kind of winding occurs ahead and behind the polymerase?

Back 24

Ahead: unwinding leading to positive supercoils

Behind: rewinding leading to negative supercoils

Front 25

What strand is the RNA strand complementary to? Which strand is the RNA the same as?

Back 25

Complementary - DNA template strand

Same - DNA nontemplate

Front 26

In RNA polymerase, what is the function of the sigma subunit?

Back 26

To recognize the promoter sequences

Front 27

In RNA polymerase, what is the core catalytic core?

Back 27

Consists of 2 alpha, beta, beta prime, and omega

Front 28

In bacteria systems, how does RNA polymerase know when to terminate?

Back 28

Destabilization by AU hybrid and self-complementary that forms a harpin structure

Front 29

p (ro) dependent

Back 29

The p protein has RNA-DNA helicase activity, but lacks the A-rich region.

Front 30

Polycistronic

Back 30

More than one protein coding sequence on the same RNA.

One RNA, multiple genes.

Occurs in prokaryotes.

Front 31

Operon

Back 31

Single promoter regulates several genes.

Front 32

What does Pol I transcribe?

Back 32

rRNA

Front 33

What does Pol II transcribe?

Back 33

mRNA, snRNA and miRNA

Front 34

What does Pol III transcribe?

Back 34

tRNA
5S rRNA

Front 35

In eukaryote, how does RNA polymerase II bind to the promoter?

Back 35

Basal Transcription Factors (TFIIB,F, E, H, A, D) bind to promoter and recruit RNA polymerase.

Front 36

In eukaryote, what unwinds the DNA? How does it change the complex?

Back 36

TFIIH turns the complex from closed to open.

Front 37

In eukaryote, which TF are lost after promoter clearance?

Back 37

TFIIE and TFIIH

Front 38

In eukaryote, what is the function of the elongation factors?

Back 38

Prevent pausing and dissociation of the RNA polymerase.

Front 39

In eukaryote, what needs to be phosphorylated for initiation?

Back 39

CTD or the C terminal domain of the RNA polymerase II

Front 40

Replicase

Back 40

Copy RNA to RNA; important in viruses that carry an RNA genome.

Front 41

What class of viruses used reverse transcriptase activity?

Back 41

Retroviruses

Front 42

How does postranscriptional processing differ in eukaryotes and prokaryotes?

Back 42

In prokaryotes, nascent mRNA is translated in proteins at the same time as transcription.

In eukaryotes, the primary transcript has to be processed before it can be exported and translated to protein.

Front 43

What is the advantage of postranscriptional processing?

Back 43

It offers greater regulation of gene expression.

For example, temporal and tissue specific expression.

Front 44

In general, what postranscriptional processing entail?

Back 44

5 Cap
Splicing
Polyadenylation

Front 45

What is the function of the 5 Cap?

Back 45

Enhances stability
Roles in processing
Roles in translation

Front 46

What is the cap and how is it linked?

Back 46

7-Methylgaunosine linked by 5', 5' Triphosphate linkage

Front 47

What is the function of Poly(A) tail?

Back 47

Roles in stability
Roles in translation

Front 48

How does termination occur eukaryote transcription?

Back 48

An enzyme complex recognizes polyadenylation signal, cleaves the RNA transcript(endonuclease activity of the enzyme complex) and adds

Front 49

Polyadenylation signal

Back 49

Facilitates termination with a sequence AAUAAA.

Front 50

Exon

Back 50

Coding (expressed) sequence

Front 51

Introns

Back 51

Noncoding intervening sequence which are removed during splicing.

Front 52

What kind of reaction does splicing entail?

Back 52

Transesterification (2) whereby the phosphodiester bond is attached by hydroxyl group (nucleophile)

Absolute conservation of phosphate

Front 53

In group II intron splicing, what is the nucleophile in the 1st transestrification?

Back 53

2' OH of a specific adenosine

Front 54

In group II intron splicing, what is the nucleophile in the 2nd transestrification?

Back 54

3' OH of the 5' exon

Front 55

How many phosphodiester bonds does the adenosine in the lariat intermediate have?

Back 55

3

Front 56

What is the difference between group II and nuclear mRNA?

Back 56

Although the mechanism are the same, Group II proceed through RNA based catalysis but nuclear mRNA requires proteins.

Front 57

snRNPs

Back 57

snRNA complexed with proteins
(U1-U6)

Front 58

How do snRNPs function?

Back 58

Recognize splice site by RNA-RNA interactions.

Front 59

Where are most of the conserved sequences in splice sites?

Back 59

Introns

Front 60

What splice site does U1 recognize?

Back 60

Forms complementary RNA interactions with the 5' splice site.

Front 61

What splice site does U2 recognize?

Back 61

Complementary to the branch point and bulges out the A nucleophile through its RNA-RNA interactions

Front 62

What is ATP hydrolysis used for in splicing?

Back 62

Not the transesterification reactions, but rather used by accessory protiens to drive the conformational required to unwind the DNA.

Front 63

Advantage of introns

Back 63

Harbor regulatory function
Alternative RNA processing producing multiple mRNAs from a single primary transcript

Front 64

How are rRNA processed?

Back 64

By cleavage(nuclease) instead of splicing as well as methylation.

Front 65

Where does rRNA processing occur in vertebrates?

Back 65

Nucleoulus

Front 66

SnoRNA

Back 66

Small nucleolar RNAs that are involved in vertebratee rRNA processing.

Recognize cleavage sites with RNA-RNA interactions.

Front 67

What enzyme carries out tRNA processing at the 5' end?

Back 67

RNase P (one of the first enzymes shown to be RNA-based catalysis)

Front 68

What critical intron needs to be spliced out in the intermediate tRNA?

Back 68

An intron interrupts the anticodon loop.

Front 69

By what mechanism does the tRNA splicing occur?

Back 69

Through an endonuclease cleavage and requires phosphate (nonconserved) from ATP

Front 70

What is activated and what acts as a nucleophile in tRNA splcing?

Back 70

The 5' OH is activated by AMP (from ATP releasing PPi).

The 3' OH acts as an nucleophile forming a 3' to 5' phosphodiester bond.

Front 71

In group I intron splicing, what acts as an nucleophile in the first tranestrification?

Back 71

The 3 'OH of detached guanosine

Front 72

In group I intron splicing, what acts as an nucleophile in the second transestricification?

Back 72

The 3' OH of the 5' exon.

Front 73

In group I intron splicing, where does the independent guanosine end up at the end of splicing?

Back 73

The guanosine ends up at the head of the intron intermediate.

Front 74

Ribozymes definition and functin

Back 74

RNA molecules wit catalytic activities but do not necessary need to have turnover.

Common in phosphodiester cleavage and transestrification.