Macromolecular Synthesis Flashcards
1
Q
Structure of DNA
A
- nucleotides: one pentose sugar, base (ATGC), phosphate
- pentose deoxyribose
- AT base pair with 2 hydrogen bonds
- GC base pair with 3 hydrogen bonds
- C and T Are pyrimidine bases (one ring sugar)
- G and A are purine bases (two ring sugar)
- DNA stability encouraged by stacking interactions (VDWF), hydrophilic outside (phosphates), hydrophobic center (base pairs)
- instability caused by electrostatic repulsion between charged phosphates; mitigated by histones
2
Q
Structure of RNA
A
- ribose (pentose), base (AUGC) and phosphate
3
Q
The cell cycle stages and regulation
A
- cell cycle regulated by checkpoints: cyclin, cyclin-dependent kinases to ensure environment is favourable
- G1: cell checks if environment is stable and if cell is big enough
- S: cell growth and development (duplication of genetic material)
- G2: checks all DNA is replicated, and if enviroment is stable
- M: mitosis
4
Q
DNA replication
A
1) DNA helicase unwinds DNA to form the replication fork, single strand binding protein prevents reannealing
2) Leading strand is replicated via DNA polymerase I (3’ to 5’), 3’OH group nucleophilic attacks 5’P on adjacent nucelotide, catalysed bond formation by pyrophosphatase- exergonic reaction
3) lagging strand (anti-parallel) replicated in discontinuous fashion (5’ to 3’= unfavourable), short RNA primers bind and DNA polymerase III catalyses elongation in short bursts (Okazaki fragments). DNA polymerase I exonuclease activitu removes primers and adds DNA. DNA ligase stitches fragments together
5
Q
The nucleosome and chromatosome
A
- the nucleosome: x2 of each H2A, H2B, H3, H4 (octamer), 146bp of DNA wrapped around 1.75 turns
- chromatosome: nucelosome + H1 histone
- key role in reducing repulsion between DNA and condensation of genetic material
6
Q
mRNA synthesis
A
- occurs in 5’ to 3’ orientation (opposite to DNA)
- TFIID binds to TATA box upstream of core promoter using TBP
- TFIIA & B bind
- TFIIF binds with RNAPII
- TFIIE & H then bind
- Zn finger motifs in TF help bind to DNA in stable manner
- RNAPII is phopshorylated at C terminus (rich in serine and threonine), TFs dissociate and gene is transcribed
- protein complex which cleaves at terminating region and is involved with poly(A) tail associated with C terminus of RNAPII
7
Q
regulation of mRNA synthesis: enhancers
A
- activator proteins bind to enhancer regions upstream of core promoter
- bind and interact with TF on the initiation complex, stimulating transcription
8
Q
Regulation of mRNA synthesis: repressors
A
- repressor proteins bind upstream of core promoter in a silencing region
- this silencing region typically overlaps with enhancer region, meaning activator proteins cannot bind and transcription is repressed
9
Q
Regulation on mRNA synthesis: co-activators
A
- histone modifcation to make DNA more readily accessible to RNAPII and associated basal TFs
- N terminus of histone proteins can be modified, usually on R group of lysine (nitrogen rich)
- acetylation, methylation, phosphorylation
10
Q
Regulation of mRNA synthesis: co-repressors
A
- histone deacetylase activity
- removes modification on histones so they aren’t as readily accessible
11
Q
Post transcriptional modification of mRNA: splicing
A
- splicing removes introns so only coding exons are on mature mRNA
- snRNA (small nuclear RNAs) and proteins come together to form the spliceosome, through series of transesterifcation reactions
- the 2’OH of the branch site in the intron nucleophilically attacks the 5’P at the donor site, forming a lariat structure
- the 3’OH on the exon at the previous donor site attacks the 5’P at the acceptor site
- phosphodiester bond formed and intron removed
12
Q
Post- transcriptional modification of mRNA: 5’ capping
A
- 7 methylguanine cap added to 5’ end
- prevents degradation
- acts as a protein binding site to allow for export of out the nucleus into the cytosol
13
Q
Post-transcriptional modification of mRNA: poly(A) tail
A
- poly(A) tail added to 3’ end of mRNA to prevent degradation
14
Q
Different types of RNA: siRNA
A
- small or short interfering RNAs
- complementary sequences to mRNA
- form part of the protein- containing RISC complex (RNA induced silencing complex) whcih base pair to the mRNA and induce mRNA degradation
15
Q
Different types of RNA: miRNA
A
- fragments of RNA which move from nucleus to cytosol
- processed by Dicer in the cytosol to give rise to mature miRNA to form part of the RISC complex
- if binding to mRNA is complete this can cause degradation
- if binding to mRNA is incomplete this causes inability for the translational machinery to bind
16
Q
Structure of tRNA
A
Anticodon loop
Anticodon stem
Acceptor stem
CCA 3’OH terminus which binds to amino acid
17
Q
Charging of tRNA
A
- aminoacyl tRNA synthetases catalyse charging
- tRNA acceptor can be an ‘iso acceptor’ for wobble base pairs
1) amino acid is adenylated
2) charging: transfer of adenylated amino acid on the tRNA involves binding of hydroxy group at the CCA 3’OH end and releases AMP
18
Q
Translation
A
- eIF1,2,3,4 bind to the small ribosomal subunit which is bound to cahrged aminoacyl tRNA, with GTP
- small subunit scans mRNA until reach start codon
- large subunit (60s) binds (ribozyme with peptidyl transferase activity)
- new charged tRNA enters the A site, and clamo slides along, old tRNA moves to P site
- E site is exit site
- adjacent amino acids condense and form peptide bond, catalysed by ribozyme
- continue until meet STOP codon (no charged tRNA for this)
19
Q
Protein targeting by the ER
A
- as N terminus of synthesised protein is exposed in cytoplasm, if has signal sequence, this is recognised by signal recognition particle (complex of proteins and non coding RNA)
- SRP binds to N terminus and to SRP receptor on RER, which is coupled to translocon
- GTP is hydrolysed, causing conformational change and dissociation of SRP
- translocon opens, peptide moves through
- signal peptidase clips N terminal signal sequence
- peptide folds in ER lumen and is moved by secretory vesicles to the golgi (where modified by glycosylation etc)
20
Q
Glycosylation of proteins
A
- N linked: occurs in ER, attached to amine N of asparagine
- O linked: less common, attached to hydroxyl groups of Ser, Thr, hydroxylysine (less common)
21
Q
Protein targeting for secretion or insertion into the plasma membrane
A
- proteins packaged into secretory vesicles from the RER
- move to the golgi apparatus, where glycosylation (O linked) can occur
- vesicles bud off from the golgi, where can be targeted for degradation in lysosomes, insertion into plasma membrane or secretion
22
Q
Proteolytic processing of insulin
A
- human insulin gene has 3 exons and 2 introns which are processed to form mature insulin in the cytosol
- hydrophobic signal sequence (N terminal leader sequence) causes binding of SRP and movement onto RER, so transcription can continue in the lumen
- introns are spliced following transcription of mRNA, forming preproinsulin
- N terminal leader sequence is cleaved (24 residue) is cleaved post-translation, forming proinsulin in the RER lumen (by signal peptidase)
- proinsulin is transported from the RER to the golgi
- in the golgi, proinsulin is then cleaved again to form insulin A and insulin B, with a connecting peptide (which retains the conformation of A and B); A and B are then connected by disulfide bridges
- secretory granules are secreted by exocytosis releasing mature insulin and C peptide
23
Q
The proteasome pathway for degradation
A
- breakdown of skeletal muscle during starvation
- predominately for abnormal proteins, short lived proteins (regulatory), long lived normal proteins i.e. contractile proteins in muscle, membrane proteins
- ubiquitin is constitutively expressed but can be upregulated in stress, this tags proteins for degradation
- enzyme cascade activated: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin protein ligase (E3), ubiquitin ubiquitin lovase
- proteasome degrades tagged proteins in an energy dependent process, into di and tri peptides
- peptides are transported to the lysosome via peptide transporter (PEPT1) on lysosome membrane, then subsequently degraded
24
Q
Lysosome degradation
A
- predominately for endocytosed proteins (hormones) and membrane proteins i.e insulin receptors
- lysosomes fuse with vesicles and release hydrolases/ proteases
