The Central Dogma (11-21) Flashcards
What are the base pairing rules for RNA synthesis?
C - G
A - U
How can cytosine produce uracil?
Cytosine can undergo spontaneous deamination to produce uracil
Why is the spontaneous deamination of cytosine an issue?
It can introduce mutations - DNA replication after deamination could replace a C-G base pair with U-A base pair (as C has become U)
Why is uracil not found in DNA?
In DNA any uracil is removed by uracil-DNA-glycosylase generating an abasic site, which is removed and repaired by DNA polymerases.
What are the 3 major classes of bacterial RNA?
- messenger RNA
- ribosomal RNA
- transfer RNA
What is the function of the 5’ promoter in bacterial transcription?
To attract and bind the RNA polymerase
Why does bacterial transcription and translation occur simultaneously?
Bacteria don’t have nuclei
What is the bacterial core RNA polymerase composed of?
α, β, β’ and ω subunits
What is the function of the sigma factor?
To bind to the core polymerase and direct it to a promoter - the addition of a sigma subunit converts it to a holoenzyme (complete functional enzyme)
E.coli has multiple sigma subunits which recognise different promoters - provide specificity
During bacterial transcription initiation why does the polymerase pull downstream DNA towards itself?
To scrunch the DNA until by chance the -10 region is open converting the closed promoter complex into an open promoter complex. Unlike the action of DNA helices it doesn’t require energy.
How does scrunching DNA during bacterial transcription affect the coiling of DNA?
downstream DNA becomes loser - negative supercoiling
upstream DNA becomes tighter - positive supercoiling
Does RNA polymerase require a primer?
No
When does the sigma factor disengage?
After 10 nucleotides of RNA synthesis - sigma factor is exposed and disengages ready for elongation
During bacterial elongation what are the characteristics of RNA polymerase?
highly processive
low fidelity - many errors
What happens if RNA polymerase mis-incorporates a ribonucleotide?
It hesitates, back-tracks, removes the nucleotide and then continues
Why is a high error rate of bacterial transcription tolerated?
If the transcript encodes a protein then most of the protein will be fine but a small subpopulation might be mutant - can probably be tolerated
What is Rho (p)-independent termination?
a terminator sequence in RNA recognised - as the RNA is being formed secondary structures start building (e.g. G-C base pairing forms a hairpin loop) here RNA pauses and dissociates due to weak base pairs
What is p-dependent termination?
requires p protein to break the RNA:DNA duplex in the transcription bubble - p protein is a hexameric helicase that binds C-rich G-poor sequence in the RNA and uses helicase activity to chase RNA pol, catches and disrupts the DNA:RNA hybrid releasing the RNA.
What is refampicin?
an inhibitor of prokaryotic transcription
inhibits RNA pol by binding tightly to the RNA exit channel - affecting initiation, preventing translation
Why doesn’t RNA polymerase require energy to open the helix?
When RNA pol binds to DNA it bends the DNA duplex so it can be opened more easily.
How are eukaryotic transcripts processed before being transported to the cytosol?
The primary transcript is:
1. capped at the 5’ end
2. spliced to remove introns
3. polyadenylated at the 3’end
How many RNA polymerases are there in eukaryotes?
3 - RNA polymerase I, II, III
RNA polymerases are complex compared to bacterial, what does this suggest about their mechanism of transcription?
The RNA polymerases differ greatly so have a different mechanism transcription.
What are general transcription factors in eukaryotic transcription?
Additional proteins required by eukaryotic RNA pols for initiation.
What are promoters in eukaryotic transcription?
A region of DNA upstream from a gene where relevant proteins bind to initiate transcription.
What are TATA box promoters?
Have an upstream sequence TATAAAA between -30 and -100 from the transcriptional start.
What are the shared homology of archaeal - eukaryotic transcription components?
Overall a similar process:
1. archaea have one RNA pol similar to eukaryotic pols
2. both have internal membranes
3. both have TATA box promoters
4. both transcribe tRNAs as individuals RNAs
suggests phylogenic relationship
What is the function of the TFIID complex in eukaryotic initiation?
TFIID guides RNA Pol II to its promoters
its large, has 11 TAF (TBP associated factors) and the TATA-box binding protein (TBP)
What does the TBP subunit of TFIID do?
Binds to the TATA box - causes DNA to bend and minor groove to widen
What does bound TBP do?
Recruits other transcription factors:
TFIIA binds TBP
TFIIB binds TBP
TFIIB recruits TFIIF, RNA Pol II, TFIIE and TFIIH - assemble around the promoter forming the basal transcription apparatus
What is the function of TFIIH in eukaryotic transcription?
- is a helicase - uses energy from ATP hydrolysis to locally unwind the DNA double helix
- is a kinase - phosphorylates the c’-terminal domain (CTD) of RNA Pol II, -ve charge changes its shape, releasing from TFs to start elongation
What happens during elongation in eukaryotic transcription?
- TFIIB, E and H dissociate from basal transcription apparatus
- RNA is synthesised
- RNA Pol II progresses, freeing the promoter and TFIIDA complex for further recruitment
What is α-amantin?
A potent inhibitor of RNA Pol II - binds tightly to the active site constraining the flexibility required to translocate DNA reducing rate of RNA production
What are enhancers?
Regulatory DNA sequences that increase the transcription levels of genes - promote high level expression
work over long distances
active in a tissue-specific manner
What is the DNA looping model?
Proteins bound to a distance enhancer interact with components of the transcription initiation complex, thus looping out the intervening DNA
How do enhancers react with RNA Pol II?
Mediator proteins act as a bridge between activator proteins that bind the enhancer control elements and the non-phosphorylated CTD of RNA Pol II (initiator state).
What provides the means of turning genes ‘on’ or ‘off’?
Enhancer/silencer elements
How do enhancers allow for cell-specific control of gene expression?
Activator proteins are required to bind enhancer control elements, transcription can only be enhanced if the appropriate activators are present .
How is eukaryotic transcription regulated by histones?
Histone acetylation
Acetylation of pertruding histone tails on nucleosomes neutralises the +ve charge on lysine - loosening its reaction with -ve DNA causing the nucleosome to unwind slightly. Loose chromatin structure permits transcription.
How are RNA Pol II transcripts modified?
- 5’ end is capped by a nucleotide triphosphate + methylation
- 3’ end is trimmed and a poly-A tail is added
- introns are removed by splicing
How is the 5’ cap added during RNA processing?
The 5’ mRNA triphosphate is modified:
one phosphate group removed, diphosphate left attacks the α-phosphate of GTP forming an unusual 5’-5’ triphosphate linkage
The N-7 of the terminal guanine is methylated to form cap 0, further methylations produce caps 1 and 2.
What is the function of the 5’ cap?
The 5’ cap looks like the 3’ end of RNA - resistant to 5’ exonucleases
Prolong half life of mRNAs
Enhance translation
How is the poly-A tail added during RNA processing?
The 3’ end of the primary transcript is cleaved by a specific exonuclease downstream of the motif AAUAAA
150-250 adenylate residues derived from ATP are added by polyA polymerase
Why is the poly-A tail important?
For the stability of mRNA - the tail is shortened overtime and when its short enough the mRNA is enzymatically degraded
What are the main differences between bacterial and eukaryotic transcription/translation?
Bacterial - mRNA molecules are translated while being transcribed and are generally not modified
Eukaryotic - mRNA precursors are processed and spliced in the nucleus then transported t the cytosol for translation
What are exons?
Nucleotide sequences that remain present in mature DNA
Covalently bonded during RNA splicing
Most less than ~1000 nucleotides long, many are ~100-200 long
What are introns?
Nucleotide sequences that are removed by RNA splicing
Possession is variable: histones have no introns (assume everything else does)
More DNA is devoted to introns than exons
What is R-loop analysis?
A laboratory technique used to analyse gene organisation - distinguish introns from exons
RNA-DNA hybridisation monitored by electron microscopy
Loops show displaced DNA
What are the 4 classes of introns?
- Group I: self-splicing, found in organelles (mitochondria, chloroplasts
- Group II: self-splicing, found in fungi/plants organelles
- Spliceosome-dependent
- Nuclear tRNA
What are the conserved features of introns?
- 5’ splice site
- 3’ splice site
- branch site (group II and spliceosomal introns)
What did Thomas Cech’s work with the rDNA of Tetrahymena show about group I introns?
The Tetrahymena rDNA intron can self-splice in the absence of any protein - RNAs can have catalytic functions they can be ribozymes
How is the splicing of group I introns performed?
Two sequential tranesterfication reactions - exchanging the R’’ group of an ester with the R’ group of an alcohol
What does the co-factor do during the splicing of group I introns?
The 3’-OH of the co-factor (guanosine, GMP, GDP or GTP) acts as a nucleophile - attacks the phosphate at the 5’ splice site
What attacks the 3’ splice site during the splicing of group I introns?
The 3’-OH of the upstream exon - becomes a nucleophile that attacks the 3’ splice site phosphate
Why does the intron fold for splicing?
So the 5’ and 3’ splice sites are close - allows for efficient and accurate transesterfication reactions
How does group II intron splicing differ from group I?
Group II introns carry their own co-factor
An internal nucleophile is used - the 2’-OH of the branch site adenine attacks the 5’ splice site phosphate forming a lariat structure
What is the result of group II intron splicing?
Fusion of the upstream and downstream exons and release of the intron in its lariat form
What are maturases?
Enzymes that improve the efficiency of intron splicing
What is the intron-early hypothesis?
All 3 domain of life have introns so they must be of ancient origin - modern organisms maintain them so they must be useful/valuable
What are homing endonucleases?
Enzymes that catalyse intron mobility - move intron from one location/organism to another
Introns may be parasitic nucleic acids that code for a protein which allows them to spread selfishly
What is the role of the spliceosome in spliceosome dependant RNA splicing?
Inactive spliceosome assembles - bringing the splice sites close together
The spliceosome is then activated and provides a framework within which splicing occurs
How is the tranesterfication reaction of spliceosome dependent RNA splicing made more efficient?
The branch site of adenine sits on a bulge, due to the base-pairing between U6 snRNA, U2 snRNA and the branch site, brining it close to the 5’ splice site
What does the phosphylated C-terminal domain of the L’subunit of RNA Pol II recruit?
- capping factors
- 3’ end processing factors
- spliceosomes
How is intron removal co-ordinated with transcription?
Spliceosomes are recruited by RNA Pol II - eukaryotic cell has control over the intron even if they are parasitic
What is alternative splicing?
A mechanism that generates protein diversity - one gene is used to make more than one protein
What happens if there are mutations in RNA splicing?
Mutations can destroy splice sites or create new splice sites - exons can be skipped or mis incorporated leading to genetic diseases
What is the diamond code?
Proposed by George Gamow 1954, concluded that the genetic code is overlapping, degenerate and triplet - each amino acid fits directly into diamond shaped pockets within DNA grooves
What did Sidney Brenner propose about the genetic code?
The code is written in a non-overlapping triplet style - if it were overlapping there would be constraints in the order of bases, some combinations would be impossible
How was the triplet code experimentally determined?
In 1961 Crick and coworkers combined mutations of FC0 and showed that +3 or -3 restored the reading frame
What are the subunits of a bacterial ribosome?
50S - catalyses the formation of peptide bonds to link amino acids
30S - provides a framework where tRNAs can be matched to codons
What does an assembled bacterial ribosome consist of?
Assembled 70S ribosome -
E: tRNA exit site
P: peptide site
A: aminoacyl-tRNA site
What are Svedberg units?
Unit for sedimentation rate - how fast a particle of given size and shape ‘settles’ at the bottom of a solution
- not additive
What is the Shine-Dalgarno sequence?
A short sequence dominated by purines - recruits the ribosome
How is the bacterial ribosome S subunit kept inactive until mRNA and an initiating tRNA are available?
IF-1 blocks the A site
IF-3 prevents premature assembled of the ribosomal S and L subunit
What is the function of initiation factors in bacterial translation initiation?
To co-ordinate ribosome assembly - resulting in a ribosome positioned around a specific initiator tRNA base-paired to a start codon in the P site
How does bacterial translation elongation occur?
- EF-Tu guides the next aa-tRNA into the A site
- a peptide bonds is formed
- EF-G enters A site, ribosome moves one codon down, deacylated tRNA enters E site, dipeptide tRNA enters P site
- incoming tRNA displaces EF-G and the tRNA leaves the E site
How is bacterial translation terminated?
- a release factor binds to the STOP codon
- the peptidyl-tRNA bond is cleaved, releasing the protein
- the mRNA-ribosome complex disassembles
Why can many ribosomes travers a single bacterial mRNA?
Bacterial mRNAs are polycistronic - has multiple genes with a ribosome-binding site (S-D) required for each
What provides the peptidyl transferase activity required for the formation of peptide bonds between aa?
Ribosomes - they are a giant catalytic ribozyme
What does an assembled eukaryotic ribosome consist of?
Assembles 80S ribosome -
P: peptidyl site
A: aminoacyl-tRNA site
(no clearly defined E site)
What are the subunits of a eukaryotic ribosome?
60S and 40S
Why are eukaryotic mRNAs often circularised?
To facilitate the re-binding of ribosomes - if it falls off doesn’t have to diffused very far to rebind - efficient
Eukaryotic mRNA doesn’t have a Shine-Dalgarno ribosome binding site, what does it do instead?
The small subunit of the ribosome scans the mRNA until it finds a translational start signal
What happens after the multiple initiation factors assemble with the 40S subunit?
Binding of the charged initiator tRNA
Binding if the 5’cap of the mRNA to the S subunit
How are secondary structures on eukaryotic mRNA unwound?
The helices activity of elF4A in the initiation complex
What is the function of the kozak sequence?
A recognition sequence that signals to the ribosome that the next AUG is the initiator codon
How is eukaryotic translational elongation carried out?
- an incoming aa-tRNA enters the A site, a peptide bond is formed
- eEF2 enters the A site - ribosomes translocates by one codon, deacylated tRNA leaves
- repeats
How is eukaryotic translational termination carried out?
A single release factor, which recognises all 3 STOP codons, cleaves the peptidyl-tRNA bond, releasing the protein, ribosome disassociates
What does the antibiotic puromycin do to protein synthesis?
Leads to premature termination - after puromycin added aa can’t be - it mimics charged tRNA enters the A site and transfers to the growing chain
What does ricin do to protein synthesis?
Inhibits the translocation of elongation - protein synthesis ceases, cell dies
What is covalent bonding?
Sharing of electron pair to complete outer shell
Is there free rotation around a double covalent bond?
No - causes structure to become flat
What is cis/trans isomerism?
Stereoisomers with the same formula but functional groups in different orientation - exists when there’s restricted rotation around a molecule
- Cis: same side (less stable)
- Trans: different sides
What are 3 main points about covalent bonds?
- Covalent bonds are strong
- Give direction and shape
- Double bonds are stronger and have no rotation
What are isomers?
Molecules that have the same molecular formula but different arrangements of atoms in space
What are conformers?
Different arrangements of molecules due to rotating around particular bonds
Structures arising from bond rotation are called conformations
What is stereoisomerism?
Isomers that differ in the orientation of groups attached to a chiral carbon (C with 4 different groups) - non superimposable
What are the different glucose polymers and their properties?
Glycogen - fast breakdown of energy
Cellulose - fibre for structure
Starch - (amylose and amylopectin) breakdown for energy
How are macromolecules stabilised?
Non-covalent bonds stabilisation rotations around covalent bonds
Why are non-covalent interactions important in biology?
Non covalent bonds are:
individually weak
collectively strong
give flexibility
Important because e.g. membranes must be semi fluid, DNA strands must come apart
How do polar covalent bonds form?
The unequal sharing of electrons due to electronegativity - the power of an atom to attract electrons to itself
What are hydrogen bonds?
Dipole - dipole interaction
The electrostatic attraction between H bonded to a highly electronegative atom with a lone pair of electrons
Angled H bonds are weaker
What are salt bridges?
Bonds between oppositely charges residues - charged amino acids make salt bridges (weak)
What are Van der Waal’s bonds?
Weak electrostatic interaction between induces dipoles, induced by proximity - Van der Waal’s distance is the optimum distance, too close leads to repulsion
What is at the N and C terminal of polypeptide chains?
N terminal: NH3+
C terminal: COO-
Are amino acids stereoisomers?
Yes - the C has 4 different groups attached so its chiral
Forms two steriosimers D and L, only L is found in humans
What is a zwitterion?
A molecule that contains an equal number of +ve and -ve function groups
At neutral pH amino acids are zwitterions - have no net charge (NH3+ and COO- balance)
What is the physiological form of amino acids at high and low pH?
Low pH: positive charge, NH3+ and COOH - high conc of H+ pushes COO- to accept H+
High pH: negative charge, NH2 and COO-, low conc of H+ pushes NH3+ to loose H+
both forms can coexist
What is the primary structure of proteins?
The sequence of amino acids
What is the secondary structure of proteins?
The conformation of the protein backbone determined by hydrogen bonding between backbone atoms (no R groups)
Why are peptide bonds planar?
Peptide bonds are partially double bonded
Leads to cis/trans isomers - cis leads to steric clashes so all are trans other then proline (due to its unusual ring structure)
What are Phi and Psi angles?
Torsion (twisting) angles of the poly peptide backbone
Phi: right-handed rotation around Cα-N bond
Psi: right-handed rotation around Cα-C bond
How is the α-helix secondary structure formed?
Backbone twists itself (right-handed) into a helix - C=O H bonds with N-H 4aa down
Side chains extend outwards and are not involved
DNA binding proteins are often α-helix to fit in the major grooves of DNA
Describe the β-strand secondary structure:
β-strands hydrogen bonds with each other to form β-pleated sheets with alternating side chains
Parallel: one aa H bonds to 2 aa in adjacent strands
Anti-parallel: one aa H bonds to 1 aa in the adjacent strand
What holds the protein tertiary structure together?
Salt bridges: interaction between opposite charges
Hydrogen bonds
Hydrophobic interactions: hydrophobic molecules clump together to exclude water
Disulphide bonds: covalent
Van der Waal’s forces: tight packing of side chains, no holes on proteins
What is a motif?
Common elements that fit together
e.g. the combination of secondary structure elements ββ
What is a protein domain?
A region of the polypeptide chain that folds independently forming a compact 3D structure made up of motifs
They give stability to aa chains and help support the extracellular matrix
What is the quaternary structure of proteins?
Proteins made up of more than one polypeptide chain
e.g collagen - tight triple helix
What is the conclusion for Anfinsen’s experiment?
Proteins adapt their native structure (fold) spontaneously
What is the order of protein folding?
- Nucleation: hydrophobia regions condense (hydrophobic collapse) - short stretches of secondary structure
- Aggregation: motifs, domains - extensive secondary structure
- Compaction: tertiary structure is obtained
What are chaperonins?
Enzymes that encase proteins are allow them to fold to their native shape inside
How does the vast machinery in cells help to fold proteins?
The machinery doesn’t interfere with the inherent ability of a protein to fold (spontaneous) it just speed up the process and prevents incorrect bonding of side chains
What is sickle cell anaemia?
A disease caused by protein misfolding - haemoglobin aggregates to form long chains that distort the shape of the cell
