Molecular biology Flashcards
1
Q
DNA Polymerase 1
A
- prokaryotes
- gene A
- replication, recombination and repair
1. 5’-3’ DNA dependent DNA polymerase ⇒ replace primer
2. 3’-5’ exonuclease ⇒ proof reading
2. 5’-3’ RNA dependent DNA polymerase - gap filling following DNA replication, repair and recombination
2
Q
DNA Polymerase alpha
A
- eukaryotes
1. 5’-3’ DNA dependent RNA polymerase
2. Primase activity
3
Q
DNA Polymerase 2
A
- prokaryotes
- gene B
- not DNA dependent
- DNA proofreading and repair
- mitochondrial DNA synthesis
4
Q
DNA Polymerase beta
A
- eukaryotes
- repair
5
Q
DNA Polymerase 3
A
- prokaryotes
- gene B
- 5’-3’ DNA dependent DNA polumerase
- 3’-5’ exonuclease
- subunits:
- alpha: polymerase
- epsilon: 3’-5’ exonuclease
- theta: assist epsilon
- Associated accessory proteins:
- beta x2: sliding clamp increase processibility
- tao: dimerise 2 core proteins ⇒ tethring
- gamma
- delta
- delta’
6
Q
DNA Polymerase epsilon
A
- eukaryotes
1. 5’-3’ DNA dependent DNA polymerase
2. synthesis of leading strand
7
Q
DNA A Protein: Initator
A
- prokaryotes
- binds Ori sequence
- unwinds DNA
- load Helicase
- 150 to 250 bp
- ATP dependent
8
Q
DNA polymerase gamma
A
- eukaryotes
1. DNA dependent DNA polymerase
2. Synthesis of Mt DNA
9
Q
DNA B: Helicase
A
- prokaryotes
- DNA B is not an active helicase when in the P/dnaB/O complex
- P is removed by heat shock proteins (dnaK, dnaJ, GrpE)
10
Q
DNA Polymerase delta
A
- eukaryotes
1. 5’-3’ DNA dependent DNA polymerase
2. synthesis of lagging stand
11
Q
DNA C: Deliver and loads helicase
A
- prokaryotes
- stabilizes DNA B ⇒ DNA C leaves the complex
- binding of C to B is ATP dependent
12
Q
ORC
A
- eukaryotes
- ORC= origine replication compkex
- binds a 11 bp sequence called origin replication element ORE, fond in ARS
- DUE= DNA unwinding element
13
Q
SSB
A
- prokaryotes
- stabilizes unwound DNA
14
Q
RPA
A
- eukaryotes
- replication protein A
15
Q
DNA G
A
- prokaryotes
- prime DNA synthesis
16
Q
RNAase
A
- eukaryotes
- eukaryotic polymerase don’t have exonuclease activity
17
Q
Gyrase
A
- prokaryotes
- negatively supercoil DNA (topoisomerase2)
18
Q
RNAase H
A
- both prokaryotes and eukaryotes
- 3’-5’ exonuclease
- remove RNA primer
19
Q
DNA ligase
A
- both prokaryotes and eukaryotes
- ligates the okasaki fragments of the lagging stand ⇒ ATPase
20
Q
Prokaryotic transcription
A
- RNA transcript ⇒ mRNA
- RNA can be polycitronic ⇒ encode for new proteins
- transriptic + translation simultaneously and same place
- RNA polymerase enoguh for transcription
- TATA box closer to coding part of gene
- 1 RNA polymerase
- shorter life time of mRNA
- RNA polymerase slides along double helix DNA until reaches promoter sequence
- promoter sequence recognized by sigma factor
- once RNA pol. reaches promoter ⇒ it latches on
- helix is opened in front of it and polymerization begins
- sigma factor is released
21
Q
Eukariotic transcription
A
- RNA transcript is modified
- mRNA only encodes 1 protein (monocitronic)
- transcription happens before, in the nucleus ⇒ translation in cytosol
- RNA polymerase requires transcription factors
- TATA box further from coding part of gene
- 3 RNA polymerase
- longer lifetime of mRNA because of cap/tail
22
Q
RNA polymerase
A
- prokaryotes
1. alpha: recognise regulatory factors
2. beta: polymerase activity, binds Mg2+ ions and composes the catalytic subunit
3. beta’: binds DNA template
4. w: unknown function
5. sigma: enables the core enzyme to recognize and bind the promoter region to form the pre initiation complex PIC
6. 30-50bp/min
7. no proof reading
23
Q
RNA Polymerase 1
A
- eukaryotes
1. forms rRNA
2. found in the nucleolus
3. insensitive to alpha-amantinin
24
Q
RNA Polymerase 2
A
- eukaryotes
1. mRNA
2. IncRNA
3. miRNA
4. SnRNA
5. found in nucleus
6. high sensitivity to alpha-amantinin
25
RNA Polymerase 3
- eukaryotes
1. tRNA
2. 5s rRNA
3. intermediate sensitivity to alpha-amantinin
26
Prokaryotic RNA Polymerase complex
- promoter sequence
- sigma binds promoter sequence
- upstream from transcription start site, we have a consensus sequence to which RNA polymerase binds to form the closed complex
- more proximal to transcription start site⇒ TATA box
- it binds to transcription factor and therefor indicates where transcription should be initiated from
27
Eukaryotic RNA Polymerase complex
- TATA box bind to TBP (subunit of TFIID)
- TAFs bind downstream promoter elements DPE and initiator sequence INR
- Caat box
* upstream cis-acting element
* binds with CTF
* increase the frequency of transcription
- GC box
* upstream cis-acting element
* bind Sp1
* increase the frequency of transcription
- TFIID⇒ complex consisting of TBP and 14 TAFs
- TFIIA,B,D ⇒ transcription initiation site ⇒ F, E, H
28
Spliceosome
- macromolecular complex responsible for precursor mRNA splicing
- consists of:
* U1: within the snRNP complex binds first by base pairing to the 5' exon - intron boundary
* U2: within snRNP complex, binds to branch site ⇒ exposes nucleophilic A residue
* U4/U5/U6: within snRNP complex mediates ATP-dependent protein-mediated unwinding that results in disruption of the base paired U4-U6 complex with the release of U4. U6 is then able to interact with U2 and then U1
* U5: enhances elignment
29
Splicing
- starts with a cut at 5' junction between intron and exon. Done by nucleophile attack from adenine just upstream from 3' end
- the free 5' forms a loop with adenine. ⇒ 5'-2' phosphodiester bond
- second cut at junction of intron and 3' exon. The 3'-OH of upstream exon attacks the 5' phosphate of downstream exon-intron junction ⇒ loop is released ⇒ 5' and 3' extremities of the eons are ligated to form a continuous segment
30
tRNA
- 3 loops
- 4 arms
- nucleotide derivates are present like iodine, pseudouridine, dihydrouridine
- phosphate at 5' end
- CCA sequence at 3' end where AA are bound
- part containing 3' and 5' arm: acceptor arm
- D-loop⇒ aminoacyl-tRNA-synthase bound
- anticodon loop ⇒ anticodon
- T-loop ⇒ binding site for ribosome
- recognition + attachement:
* AA + ATP⇒ aminoacyl-AMP + PPi
* Aminoacyl-AMP + tRNA ⇒ aminoacyl-tRNA + AMP
- A site: binds aminoacyl-tRNA
- P site: occupied by peptide-tRNA
- E site: occupied by empty tRNA
31
rRNA
- large 60s subunit (3RNA + 49proteins)
| - small 40s subunit (1RNA + 33 proteins)
32
miRNA
- pri-miRNA are 5'-capped and 3'-polyadenylated
33
snRNA
- U1, U2, U4, U5, U6 are involved in intron removal and processing of mRNA precursors into mRNA
- U7 nRNA involved in production of correct 3' ends of histone mRNA - which lacks a poly(A) tail
34
siRNA
- RNA silencing and post transcriptional regulation of gene expression by: cleavage of mRNA strand, destabilization of mRNA through shortening of poly A tail, less efficient translation of mRNA into proteins by ribosomes
35
IF-1
- prokaryotes
| - complex with small subunit
36
IF-2
- prokaryotes
- binds to GTP, it attaches to the initiator aminoacyl-tRNA ⇒ joins the small subunit and the mRNA ⇒ it goes directly into P-site where binds to AUG
- when binding of large subunit ⇒ hydrolysis of GTP on IF-2
- IF-1 and 2 are released
- the 2 subunits unite ⇒ elongation begins
37
IF-3
- prokaryotes
- complex with small subunit
- when released ⇒ binding of large subunit
38
eIF-2
- eukaryotes
- binds AA-tRNA to 5'-cep of mRNA
- GTP binding protein involved un bringing the initiation or tRNA to the P site of the pre-initiation complex
- formation of 43S pre-initiation complex
39
eIF-2a
- eukaryotes
- is phosphorylated by at least four different protein kinases (HCR, PKR, PERK, GCN2)
- binds tightly to and inactivates GTP-GDP recycling protein eIF-2b⇒ prevent formation of 43s pre initiation complex and blocking protein synthesis
40
eIF-3
- eukaryotes
- eIF-3 and eIF-1a bind to newly dissociated 40s ribosomal subunit. ⇒ delays its reallocation with 60s subunit and allows other transition initiation factors to associate with the 40s subunit
- binds with high affinity to 4G component of 4F, and it links this complex to 40s ribosomal subunit
41
eIF-4
- eukaryotes
- eIF-4a
- eIF-4g: interacts with eIF3; poly A tail binging
- eIF-4e: rate limiting step that binds to 5'cap
- eIF-4b: reduces complex secondary structure of 5' end of mRNA through its ATP- dependent helices activity
- eIF-4bp: inhibit complex assembly
- eIF-4 a+g+e⇒4f
42
EF-TU
- prokaryotes
| - elongation factor thermo-unstable mediates the entry of aminoacyl-tRNA to the entry A site
43
EF-TS
- prokaryotes
| - serves as guanine exchange factor for EF-TU, catalyzing the release of GDP from EF-TU
44
EF-G
- prokaryotes
- catalyses the translocation of the tRNA and the mRNA down the ribosome at the end of each round of polypeptide elongation
45
eEF-1a
- eukaryotes
- formas a ternary complex with GTP and the entering aminoacyl-tRNA.
- complex allows the correct AA-tRNA to enter A-site with the release of EF1A-GDP and phosphate
- GTP hydrolysis is catalysed by an active site on ribosome; hydrolysis induces a conformational change in the ribosome concomitantly increasing affinity for tRNA
46
eEF-1b
- eukaryotes
| - acts as guanine exchange factor for alpha, and catalyses the release of GDP from alpha
47
eEF-2
- eukaryotes
- binds to and displaces the peptide tRNA from the A site to the P site
- in turn the deacetylated tRNA is on the E site from which it leaves the ribosome
- EF2-GTP complex is hydrolyzed to EF2-GDP, effectively moving the mRNA forward by one codon and leaving the A site open for occupancy by another ternary complex of AA tRNA-EF1a-GTP and another cycle of elongation
48
RF-1
- prokaryotes
- termination factor
- trigger hydrolysis of ester bond in peptide-tRNA + release of new protein
- recognise stop codon resides in A site
- bound by complex consisting of RF3-GTP
- complex + peptide transferase promotes hydrolysis of bond between peptide and tRNA occupying P site
- thus a water molecule rather than amino acid is added. this hydrolysis releases the protein and tRNA from P site
49
RF-2
- prokaryotes
| - trigger hydrolysis of ester bond in peptide-tRNA + release of new protein
50
RF-3
- prokaryotes
- catalyses release of RF1 and 2
- bound to GTP
51
eRF in eukaryotes
1. primary transcript has 20 nucleotides cleaved downstream from recognition sequence
2. enzyme polyA polymerase adds poly A tail ⇒ protects 3'end from endonuclease attack. + involved in transport of mRNA to cytoplasm
52
Actinomycin D
- intercalated between C and G pairs in DNA
| - inhibits transcription, elongation in prokaryotes and eukaryotes
53
Rifampicin
- binds to beta subunit of the prokaryotes RNAP
| - blocks promoter clearance
54
Alpha-amatinin
- blocks translocation of RNA polymerase during phosphodiester bond formation
55
Chloramphenicol
- inhibitor of translation
