Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
73 Cards in this Set
- Front
- Back
|
What is the importance of Pyrophosphatase in DNA synthesis?
|
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.
|
|
|
What are the substrate requirements for DNA pol?
|
Template, primer, 3'-OH end
|
|
|
What do you need to know about Catalytic site - DNA pol, "palm" domain
|
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).
|
|
|
What do you need to know about DNA pol, "finger" domain
|
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.
|
|
|
What is the accuracy of DNA pol?
|
an error occurs once every 10,000 bases. However, mistakes are only passed on in one in every 10 billion bases; Proof reading exonucleases.
|
|
|
How does proof reading work?
|
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.
|
|
|
How are RNA primers removed?
|
RNA primers are removed by Pol 1’s 5’-3’ exonuclease function, nicks are sealed by DNA ligase.
|
|
|
How long are the primers synthesised by DnaG primase?
|
11+/- 1 nt. Primase probably associates and dissociated with the formation of each primer.
|
|
|
How big are okazaki fragments?
|
In bacteria they are 1000-2000 nt long, in eukaryotes they are 100-400 nt.
|
|
|
Describe DNA helicase:
|
DNA helicase unwind DNA at the replication fork, they are processive and require loading onto DNA.
|
|
|
How are super coils removed?
|
Topoisomerases
|
|
|
The replisome contains?
|
two DNA pol 3 enzymes, and slide clamp (and clamp loader)
|
|
|
In which direction does the replisome move.
|
As a single unit in the 5’-3’ direction along the leading strand, the lagging strand template loops around.
|
|
|
How is DNA replicated with high fidelity?
|
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. |
|
|
What is the importance of telomerase?
|
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.
|
|
|
What is the basis of DNA finger printing?
|
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.
|
|
|
How do Short Tandem Repeats arise?
|
Through replication slippage.
|
|
|
What makes up the DNA?
|
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). |
|
|
What protein complex carries out splicing?
|
splicesome
|
|
|
what are the locations and product of RNA pol 1?
|
Located in the nucleolus. Product is rRNA(ribosomal RNA).
|
|
|
what are the locations and product of RNA pol 2?
|
Located in the nucleoplasm. Products are mRNA, sno RNA and some sn RNA.
|
|
|
what are the locations and product of RNA pol 3?
|
Located in the nucleoplasm. Products are tRNA, 5s RNA, some snRNA and other small RNAs
|
|
|
What is snoRNA?
|
Small nucleolar RNA; guides the chemical modification of mRNA, tRNA and snRNAs.
|
|
|
What is snRNA?
|
Small nuclear RNA; about 150 nt, is involved in the processing of pre-mRNA in the nucleus.
|
|
|
Examples of DNA dependent- RNA polymerase.
|
RNA pol I, RNA pol II, RNA pol III
|
|
|
What are the steps in a PCR reaction?
|
denature DNA at 95 C, add primers and anneal at 50-60 C, extend chains with Taq Pol and ndtps at 72C. REPEAT STEPS
|
|
|
What are the primer design rules?
|
• 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 |
|
|
How is Analyzing expression by sequencing (RNAseq) achieved?
|
Isolate RNA, RNA to cDNA, Fragment cDNA and make library, Sequence, ‘Map’ Sequence reads
|
|
|
Describe the formation of cDNA.
|
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.
|
|
|
What occurs to the CTD of RNA pol after transcription initiation?
|
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.
|
|
|
What phosphorylates the serine 5 residue on the CTD of RNA pol II?
|
TFIIH
|
|
|
What phosphorylates the serine 2 residue on the CTD of RNA pol II?
|
P-TEFb
|
|
|
What post-translational modifications does RNA pol II do?
|
(Introns spliced out by splicesome)
Addition of poly A tail Addition of 5' cap |
|
|
How is the 5' cap added?
|
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.
|
|
|
Transcription regulation occurs?
|
Initiation, transcriptional factors, chromatin modifications
|
|
|
What is the core promotor?
|
The region of a gene where the general transcription factors and RNA pol bind. Formation of the pre-initiation complex occurs here (PIC).
|
|
|
What is TBP?
|
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. |
|
|
Describe the formation of PIC.
|
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.
|
|
|
What are the functions of TFIIH?
|
has a helicase activity, melt DNA and phosphorylate serine 5 residue on CTD so RNA poll II can start transcription.
|
|
|
How long will mRNA that will lead into abortive transcription be?
|
up to 10 nt.
|
|
|
What is a promotor?
|
An upstream region of DNA that facilitates the transcription of a particular gene. nb different genes have different promotor sequences.
|
|
|
Cis regulatory elements are?
|
on the same strand as the transcribed region.
|
|
|
Trans acting factors are?
|
from a molecule outside the transcribed region.
|
|
|
What does the DNA binding domain do?
|
recognises the gene regulatory element (sequence motif), holds the activator domain within the vicinity of the promotor.
|
|
|
How can transcription factors act as repressors?
|
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.
|
|
|
Heterochromatin is?
|
tightly packed, highly repetitive sequences, low transcriptional activity.
|
|
|
Euchromatin is?
|
not tightly packed, gene rich, high transcription activity.
|
|
|
What is epigenetics?
|
Any potentially stable and heritable change in gene expression that occurs without a change in DNA sequence.
|
|
|
What are examples of chromatin modifications?
|
-DNA methylation
- Histone tail modification (acetylation and methylation) - Histone variants - nucleosome remodeling |
|
|
How does DNA methylation work?
|
Cytosine methylation by DNA methytransferase, it is reversible but represses transcription.
|
|
|
What are the two types of methylation irrespective of transcription regulation?
|
-Maintenance methylation = necessary to preserve DNA methylation after every cellular DNA replication cycle.
- denovo methylation = set up methylation patterns early on. |
|
|
What is the histone code?
|
Specific modifications evoke certain chromatin based functions.
Act sequentially or in combination to generate biological outcome (regulate transcription). “Writers” and “Readers”. |
|
|
What are the fundamental activities of the ribosome?
|
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. |
|
|
What causes acute amino glycosides induced deafness (AAID)?
|
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.
|
|
|
What is the Shine-Dalgarno sequence?
|
A sequence element 5-9 base upstream of the start codon in prokaryotes that signal bacterial ribosomes to bind and start translating.
|
|
|
How to eukaryotic ribosomes initiated transcription?
|
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.
|
|
|
What is the initial amino acid incorporated into prokaryotes?
|
AUG= Methianine, all bacterial methanine that act as a start sequence finder have a formyl group.
|
|
|
Describe the mechanism of formation of initiation complex on bacteria?
|
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# |
|
|
What does iron responsive element binding protein do?
|
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.
|
|
|
What do you need to know about Eukaryotic initiation complex formation?
|
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.
|
|
|
How can the amount of protein synthesised be regulated?
|
EIF4E and EIF2B (phosphorylation), miRNAs acting on 3' UTR
|
|
|
What are the two classes of gene?
|
Constitutive genes
- amount not highly regulated Inducible genes – - Expression induced or repressed, highly regulated. |
|
|
Why regulate gene expression?
|
Conserve resources, respond to internal/external environmental changes, ordered development.
|
|
|
What are non coding (ncRNA) genes encoded into?
|
tRNA and rRNA. (further processing is required to form functional RNAs)
|
|
|
What is an operon?
|
• 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. |
|
|
Describe RNA pol in bacteria.
|
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 ααββω |
|
|
What are the promotor recognition sequences on bacterial operons?
|
The sequence at -10 (the -10 element) has the consensus sequence TATAAT.
The sequence at -35 (the -35 element) has the consensus sequence TTGACA. |
|
|
How does adenosine inhibit transcription in prokaryotes?
|
Inserts pairs of GC bases into DNA, non specific inhibition.
|
|
|
How does rifpamicin inhibit transcription in prokaryotes?
|
RIF binds to the the catalytic (beta) subunit (rpoB).
|
|
|
What regulate transcription in prokaryotes?
|
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 |
|
|
What do lac Z, Y and A encode for?
|
beta galactose, permease, transacetylase
|
|
|
What is bioinformatics?
|
‘the development and application of computer science to
the analysis of large amounts of biological information. |
|
|
How is the poly-A tail added by the CTD of RNA pol?
|
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.
|
