Bioc 221 - Final Exam

Front 1

What is the importance of Pyrophosphatase in DNA synthesis?

Back 1

catalyses the conversion of pyrophosphate into two phosphates. This helps drive DNA/ RNA synthesis as the removal of pyrophosphate prevents its inhibition and makes it essentially irreversible.

Front 2

What are the substrate requirements for DNA pol?

Back 2

Template, primer, 3'-OH end

Front 3

What do you need to know about Catalytic site - DNA pol, "palm" domain

Back 3

provides correct environment for reaction (addition of dNTP to phosphate backbone) to occur, contains 2 mg 2+ ions (2 metal ion mechanism catalysis phosphotransferase reaction), Amino acid side chains e.g. Asp and discrimination amino acids (prevent addition of rNTP being incorporated).

Front 4

What do you need to know about DNA pol, "finger" domain

Back 4

Binds to incoming dNTPs, "fingers" fold over if correct base pairing occurs, twists the template strand so that only one base is in the active site.

Front 5

What is the accuracy of DNA pol?

Back 5

an error occurs once every 10,000 bases. However, mistakes are only passed on in one in every 10 billion bases; Proof reading exonucleases.

Front 6

How does proof reading work?

Back 6

When an incorrect base pair is recognized, DNA pol reverses (moves 3'-5'). The 3'-5' exonuclease activity removes the incorrect base pair. Following base excision, the polymerase can re-insert the correct base and replication can continue forwards. Not all DNA pol have an associated DNA mechanism and it can even be a different peptide.

Front 7

How are RNA primers removed?

Back 7

RNA primers are removed by Pol 1’s 5’-3’ exonuclease function, nicks are sealed by DNA ligase.

Front 8

How long are the primers synthesised by DnaG primase?

Back 8

11+/- 1 nt. Primase probably associates and dissociated with the formation of each primer.

Front 9

How big are okazaki fragments?

Back 9

In bacteria they are 1000-2000 nt long, in eukaryotes they are 100-400 nt.

Front 10

Describe DNA helicase:

Back 10

DNA helicase unwind DNA at the replication fork, they are processive and require loading onto DNA.

Front 11

How are super coils removed?

Back 11

Topoisomerases

Front 12

The replisome contains?

Back 12

two DNA pol 3 enzymes, and slide clamp (and clamp loader)

Front 13

In which direction does the replisome move.

Back 13

As a single unit in the 5’-3’ direction along the leading strand, the lagging strand template loops around.

Front 14

How is DNA replicated with high fidelity?

Back 14

1. Cells maintain balanced levels of dNTPs (dATP, dCTP, dGTP, and dTTPs)
2. Polymerase reaction has high fidelity - occurs in two stages
1. Incoming dNTP base-pairs with template while enzyme
is in open, catalytically inactive conformation,
2. Polymerisation occurs only after ‘fingers’ close around
positioned dNTP - should only occur if base is correct.
3. Proof reading ability of polymerase
4. DNA repair systems in the cell that identify errors before DNA is replicated again.

Front 15

What is the importance of telomerase?

Back 15

DNA pol cannot extend it extreme 5' end of the lagging strand as it requires a RNA primer and a free 3' OH. Removal of the RNA primer and degradation of the remaining ssDNA would result with progressively shorter DNA with each round of replication.

Front 16

What is the basis of DNA finger printing?

Back 16

Short tandem repeats or satellite dna; regions of highly repetitive DNA. Difference in how many of the repeats/length of the region is the basis for DNA fingerprinting.

Front 17

How do Short Tandem Repeats arise?

Back 17

Through replication slippage.

Front 18

What makes up the DNA?

Back 18

3% = Highly repetitive sequences eg. satelite DNA, STRs, Centromers and telomers.
45% moderately repetitive DNA eg transpoon repeats (cause of most of the variation in genome size).

Front 19

What protein complex carries out splicing?

Back 19

splicesome

Front 20

what are the locations and product of RNA pol 1?

Back 20

Located in the nucleolus. Product is rRNA(ribosomal RNA).

Front 21

what are the locations and product of RNA pol 2?

Back 21

Located in the nucleoplasm. Products are mRNA, sno RNA and some sn RNA.

Front 22

what are the locations and product of RNA pol 3?

Back 22

Located in the nucleoplasm. Products are tRNA, 5s RNA, some snRNA and other small RNAs

Front 23

What is snoRNA?

Back 23

Small nucleolar RNA; guides the chemical modification of mRNA, tRNA and snRNAs.

Front 24

What is snRNA?

Back 24

Small nuclear RNA; about 150 nt, is involved in the processing of pre-mRNA in the nucleus.

Front 25

Examples of DNA dependent- RNA polymerase.

Back 25

RNA pol I, RNA pol II, RNA pol III

Front 26

What are the steps in a PCR reaction?

Back 26

denature DNA at 95 C, add primers and anneal at 50-60 C, extend chains with Taq Pol and ndtps at 72C. REPEAT STEPS

Front 27

What are the primer design rules?

Back 27

• primers should flank the sequence of interest
• primer sequences should be unique - primers that match multiple sequences will give multiple products
• primers should be at least 15 base pairs long
• have at ~50% G/C content
• anneal at a temperature in the range of 50-65 °C (Usually higher annealing temperatures (Tm) are better i.e. more specific for your desired target)
• forward and reverse primer should anneal at approximately the same temperature

Front 28

How is Analyzing expression by sequencing (RNAseq) achieved?

Back 28

Isolate RNA, RNA to cDNA, Fragment cDNA and make library, Sequence, ‘Map’ Sequence reads

Front 29

Describe the formation of cDNA.

Back 29

formation fo RNA-DNA hybrid using poly T primer, dntps and reverse transcriptase. degradation of RNA component through RNase or alkaline solution (dissociation). DNA directed DNA polymerase makes dsDNA using 3' end as primer. Sn1 (single strand endonuclease) cuts strand in 2 making cDNA proper.

Front 30

What occurs to the CTD of RNA pol after transcription initiation?

Back 30

the serine 5 residue is phosphorylated by TF II H, after this the polymerase often pauses. P-TEFb (positive transcription-elongation factor-b) stimulates transcription by phosphorylation the serine 2 residue. The CTD is phosphorylated during and after transcription termination to facilitate a new cycle of transcription.

Front 31

What phosphorylates the serine 5 residue on the CTD of RNA pol II?

Back 31

TFIIH

Front 32

What phosphorylates the serine 2 residue on the CTD of RNA pol II?

Back 32

P-TEFb

Front 33

What post-translational modifications does RNA pol II do?

Back 33

(Introns spliced out by splicesome)
Addition of poly A tail
Addition of 5' cap

Front 34

How is the 5' cap added?

Back 34

capping enzyme (CE) recruited to the 5' triphosphate at the end of the mRNA adding an inverted guanine cap, producing 5'-Gppp. CE is a bifunctional enzyme that has both triphosphatase and guanylyltransferase activities. The 5'-Gppp cap is then methylated on N7 by a second enzyme, the cap RNA methyltransferase (RNMT), to produce 5'-m7Gppp (step 3). 5'-Gppp-capped mRNAs are stable but are weakly translated. Methylation of this cap is required for eukaryotic translation initiation factor-4E (eIF4E) binding and recruitment onto ribosomes for translation.

Front 35

Transcription regulation occurs?

Back 35

Initiation, transcriptional factors, chromatin modifications

Front 36

What is the core promotor?

Back 36

The region of a gene where the general transcription factors and RNA pol bind. Formation of the pre-initiation complex occurs here (PIC).

Front 37

What is TBP?

Back 37

TATA binding protein. Binds to DNA and causes it to bend/kink (almost 90°), potentially a signal for other proteins to bind. Joined by additional subunits (TAFs – TBP-associated factors) to form the TFIID complex.
Binds promoters without the TATA box too.

Front 38

Describe the formation of PIC.

Back 38

TBP component of TFIID bind to the core promotor, TAF recruited. TFIIA and TFIIB bind stabilising TFIID. TFIIF bind to RNA pol II and escorts it to the complex, TFIIB aids in its recruitment. TFIIE and TFIIH sequentially recruited thereby completing the complex, TFIIE "helps" TFIIH.

Front 39

What are the functions of TFIIH?

Back 39

has a helicase activity, melt DNA and phosphorylate serine 5 residue on CTD so RNA poll II can start transcription.

Front 40

How long will mRNA that will lead into abortive transcription be?

Back 40

up to 10 nt.

Front 41

What is a promotor?

Back 41

An upstream region of DNA that facilitates the transcription of a particular gene. nb different genes have different promotor sequences.

Front 42

Cis regulatory elements are?

Back 42

on the same strand as the transcribed region.

Front 43

Trans acting factors are?

Back 43

from a molecule outside the transcribed region.

Front 44

What does the DNA binding domain do?

Back 44

recognises the gene regulatory element (sequence motif), holds the activator domain within the vicinity of the promotor.

Front 45

How can transcription factors act as repressors?

Back 45

Competitive (binding of repressor to gene regulator element prevents the binding of activator), "masking" (binding the activation domain of the activator), or direct interaction with transcription factor.

Front 46

Heterochromatin is?

Back 46

tightly packed, highly repetitive sequences, low transcriptional activity.

Front 47

Euchromatin is?

Back 47

not tightly packed, gene rich, high transcription activity.

Front 48

What is epigenetics?

Back 48

Any potentially stable and heritable change in gene expression that occurs without a change in DNA sequence.

Front 49

What are examples of chromatin modifications?

Back 49

-DNA methylation
- Histone tail modification (acetylation and methylation)
- Histone variants
- nucleosome remodeling

Front 50

How does DNA methylation work?

Back 50

Cytosine methylation by DNA methytransferase, it is reversible but represses transcription.

Front 51

What are the two types of methylation irrespective of transcription regulation?

Back 51

-Maintenance methylation = necessary to preserve DNA methylation after every cellular DNA replication cycle.
- denovo methylation = set up methylation patterns early on.

Front 52

What is the histone code?

Back 52

Specific modifications evoke certain chromatin based functions.
Act sequentially or in combination to generate biological
outcome (regulate transcription).
“Writers” and “Readers”.

Front 53

What are the fundamental activities of the ribosome?

Back 53

1- Decode the mRNA
2- Maintain fidelity of information transfer and read code successively in three letter grouping (dont make mistakes in transferring between language of peptide to AA).
3- Peptide bond formation.

Front 54

What causes acute amino glycosides induced deafness (AAID)?

Back 54

previously silent mutation in gene for mitochondrial ribsosomal RNA, which makes them more sensitive to the aminoglycosides and thereby inhibited just like bacterial ribsosomes. A--> G mutation.

Front 55

What is the Shine-Dalgarno sequence?

Back 55

A sequence element 5-9 base upstream of the start codon in prokaryotes that signal bacterial ribosomes to bind and start translating.

Front 56

How to eukaryotic ribosomes initiated transcription?

Back 56

Bind to the 5' cap and roll along till the first AUG is read which it assumed to be the start codon. part of the "kozak" sequence.

Front 57

What is the initial amino acid incorporated into prokaryotes?

Back 57

AUG= Methianine, all bacterial methanine that act as a start sequence finder have a formyl group.

Front 58

Describe the mechanism of formation of initiation complex on bacteria?

Back 58

1- IF3 binds to small ribosomal subunit and also to mRNA, keeps the large subunit away from the small subunit.
2 - IF2 binds initiator tRNA (fmet-tRNA)and mRNA binds to small ribosomal subunit (IF2). IF2 blocks A-site on ribosome ensuring fmeth-tRNA gets to the P-site.
3i- Factors leave, can involve GTP hydrolysis (of IF2)
3ii- the large subunit is now allowed to associate with the small subunit as there is no IF#

Front 59

What does iron responsive element binding protein do?

Back 59

If Fe levels are low, IRE-BP will bind and stop ribosome structure scanning for start AUG. If Fe levels are high, Fe will bind to IRE-BP and it will not bind to stem loop and thus ribosome gets to AUG and Ferritin (Fe storage protein) is made.

Front 60

What do you need to know about Eukaryotic initiation complex formation?

Back 60

EIF4F (made up of EIF4A, EIF4G and EIF4E), role of this initiation factor is cap recognition and forming a complex with the 40s ribosomal subunit. Binds mRNA to small subunit. EIF2 picks up met-tRNA (EIF2.met-tRNA.GTP), delivers to the ribosome, uses GTP hydrolysis to escape as EIF2.GDP, this inactive form recycles to EIF2GTP, recycling catalysed by EIF2B. Phosphoylation of the Ser-51 residue on EIF2 results in EIF2B being trapped (high affinity), decreasing rate of initiation as levels of EIF2B decrease and recycling does not occur. Poly A binding proteins bind onto poly A tail and EIF4G bringing the cap and tail (5' end and 3' end closer together), making a circle, thought to make it much more efficient in recycling ribosomes, increasing translation efficiency.

Front 61

How can the amount of protein synthesised be regulated?

Back 61

EIF4E and EIF2B (phosphorylation), miRNAs acting on 3' UTR

Front 62

What are the two classes of gene?

Back 62

Constitutive genes
- amount not highly regulated
Inducible genes –
- Expression induced or repressed, highly
regulated.

Front 63

Why regulate gene expression?

Back 63

Conserve resources, respond to internal/external environmental changes, ordered development.

Front 64

What are non coding (ncRNA) genes encoded into?

Back 64

tRNA and rRNA. (further processing is required to form functional RNAs)

Front 65

What is an operon?

Back 65

• Set of co-transcribed genes under the control of a single regulated promoter.
• These transcripts are polycistronic (encode multiple genes/cistrons or proteins)
• Genes in operons usually have related functions but not always.

Front 66

Describe RNA pol in bacteria.

Back 66

4 subunits rpoA (two alpha subunits;enzyme assembly, promotor recognition and some binds some activators), rpoB (B subunit; catalytic centre), rpoC (B'; catalytic centre), rpoD (sigma factor = specific binding promotor)
Also ω in some RNAP therefore ααββ
ω

Front 67

What are the promotor recognition sequences on bacterial operons?

Back 67

The sequence at -10 (the -10 element) has the consensus sequence TATAAT.
The sequence at -35 (the -35 element) has the consensus sequence TTGACA.

Front 68

How does adenosine inhibit transcription in prokaryotes?

Back 68

Inserts pairs of GC bases into DNA, non specific inhibition.

Front 69

How does rifpamicin inhibit transcription in prokaryotes?

Back 69

RIF binds to the the catalytic (beta) subunit (rpoB).

Front 70

What regulate transcription in prokaryotes?

Back 70

1. Promoter strength – degree of match to the
consensus for a sigma factor.
‘strong’ or ‘weak’ promoters
2. Alternative sigma factors- active under different
conditions
3. Transcription factors - positive or negative regulation
The binding affinity of these may be modified
allosterically or covalently

Front 71

What do lac Z, Y and A encode for?

Back 71

beta galactose, permease, transacetylase

Front 72

What is bioinformatics?

Back 72

‘the development and application of computer science to
the analysis of large amounts of biological
information.

Front 73

How is the poly-A tail added by the CTD of RNA pol?

Back 73

Cleavage and poly-A signals are encoded in DNA, as mRNA exits Pol complex CTD aids in recruiting CstF and CPSF to what will become the 3' end. Further clevage factors bind and RNA is cleaved. Poly-A polymerase binds to mRNA and adds approximately 200 A's to the end of the molecule, The nucleotide precursor for these additions is ATP, and the same type of 5′-to-3′ bonds are formed as in conventional RNA synthesi. As the poly A tail is being synthesised Poly A binding proteins bind onto it and regulate the final length. Poly A binding proteins will later bind to EIF4G during protein synthesis.