Molecular Flashcards
Simple multi gene family
All genes same, proteins needed in large amounts
e.g. ribosomal RNA genes
Complex multi gene family
Genes not identical but have similar DNA sequences. Great organismal complexity
e.g. human globin genes
arises due to gene duplication over time
Molecular clock
measure of rate at which DNA sequence of a gene changes
Pseudogenes
genes that have changed so much that they have lost their function
4 in a-globin family, 1 in b-globin family
DNA topoisomerases
Remove supercoils
- Type I nicks one strand opening up DNA so replication fork can continue up strand
- Type II cuts double strand, passes other DNA strand through gap created + rejoins relieving tension in DNA
3’ -> 5’ exonuclease activity
Polymerase can remove nucleotides it has j inserted, proofreading allows correction of errors
DNA pol I and III (bacteria) and pol delta (eukaryotes) uses it
5’ -> 3’ exonuclease activity
Polymerase can remove DNA already attached to template
DNA pol I (bacteria) uses it
Leading strand replication
Bacteria - primer made w/ primase enzyme, DNA pol III replicates new strand
Eukaryotes - RNA primer extended by pol alpha, pol delta synthesises strand
Lagging strand
Must be done in sections (Okazaki fragments) as synthesis always 5’ -> 3’
Bacteria - pol III stops when reaches primer, pol I w/ 5’->3’ continues synthesis + ligase joins fragments together
Eukaryotes - pol delta + helicase push aside primer, FEN1 (endonuclease) cuts flap/branch, ligase joins fragments
Telomerase
RNA protein complex
Eukaryotes - it prevents end of chromosomes being shortened, extends parent strand by adding TTAGGG repeats so last Okazaki frag can now be primed
sequence added in prokaryotes is TTGGGG
most cells don’t have it, only expressed in stem cells + cancer cells
Genome
complete set of DNA mols possessed by a organism
Role of PCNA (proliferating cell nuclear antigen)
Acts as sliding clamp at eukaryote replication fork,
holds pol delta tightly onto DNA
Forks merge in linear DNA so no need for tight control
Initiation of replication in E.coli
2 replication forks (bidirectional) from origin of replication which has specific sequence
DnaA proteins bind close to origin forcing base pairs to break
- Pre-priming complex formed by attachment of DnaB proteins (helicase) so helix unwinds
- primosome formed by attachment of 2 primase enzymes
At replication fork in E.coli
gamma complex attaches/detaches pol III from lagging strand, beta complex holds pol III onto template
fork meets at terminator sequences, Tus proteins bind + ensure directionality so replication stopped
Chromatosome
Nucleosome + DNA + linker histone (H1)
2 types of heterochromatin
Constitutive - always tightly packed in all cells
Facultative - tightly packed only in some cells, can be opened up
Karyogram
Staining of metaphase chromosomes to create bands which cane be used for gene mapping + identifying chromosome structure
Telomere
Protects ends of chromosomes from exonuclease attack + from being mistaken as broken ends
Lac operon
5’ promoter, operator, 3 genes (lacZ, lacY, lacA)
lacZ hydrolyses lactose, lacY -> permease which allows lactose into bacteria
Kept switched off by lac repressor
No lactose - lac operon repressed, repressor binds to O preventing RNA polymerisation
Lactose present - binds to repressor so it detaches from O so pol can transcribe genes, lactose is an inducer + under neg. feedback
Cis acting genes/sequences
contain operators + promoters, only regulate DNA it is joined to and is dominant
Trans acting genes/factors
regulate genes anywhere, mostly TFs + mostly recessive
trans acting factors bind to cis acting sequences
Catabolite repression
preferential use of glucose by bacteria, so operons only active when glucose used up
uses CRP (catabolite repressor protein)
- binds to promoter near RNA pol
- cAMP binds to CRP permitting DNA binding
if high glucose, then cAMP used up (low) meaning lac operon off
Trp operon
5 genes (A,B, C, D, E), code for enzymes that synthesise tryptophan, promoter + operator
Operator binds to repressor protein stopping transcription when tryptophan present
-> Trp Operon repressed by tryptophan
Regulatory sequence of Eukaryotic class II genes
Promoter, Enhancer (has TF binding sites), intron, UTR (3’ and 5’)
eukaryotic genes large but most is non-coding
coding strand (sense) is 5’ - 3’
template strand (antisense) is 3’ - 5’
Transferrin receptor
Fe in blood binds to transferrin + enters cell via receptor, if enough intracellular Fe2+ then Tf receptor mRNA degraded
Regulated by RNA secondary structure as IRE-BP bound to 3’-UTR, it then dissociates and binds to Fe2+, RNA degraded so receptor not made
Ferritin gene
Ferritin binds to Fe2+ within cell
in low iron, IRE-BP loses its Fe2+ and binds to IRE preventing translation initiation of ferritin
Roles of RNA
rRNA (siting and catalysis) + tRNA - synthesis of proteins
snRNA - processing mRNA (splicing introns)
snoRNA - processes ribosomal RNA
catalytic RNA - self splicing introns + ribozymes
mRNA capping
Guanosine triphosphate (GTP) joins to mRNA at 5’ end
methylation at 2’ position on first 2 nucleotides + on G
-> increases stability, needed for efficient splicing
3’ cleavage + polyadenylation
CPSF binds to AAUAA, CstF binds to G/U - recruits cleaving factors + polyA polymerase
polyA tailing functionally linked to transcription
pre-mRNA splicing
Conserved sequences at 5’ and 3’ sites + branchpoint region act as signals
- cleavage at 5’ splice site, lariat formation at branchpoint
- cleavage at 3’ splice sites, intron region removed + exon ligation
Spliceosome
complex of small nuclear ribonucleoprotein particles (snRNPs)
U1, U2, U4, U5, U6 are snRNPs involved
U2+U5+U6 make up active site
Alternative splicing
Exon inclusion/exclusion can lead to altered or truncated protein products if exon has stop codon
-> isoforms can be produced from single gene, different functions
Amino acid activation
A.acids attach to tRNA 3’ acceptor arm
uses ATP -> AMP
Met codon (AUG) follows Shine-Dalgarno sequence in code (AGGAGG) -> identitifes site of initiation + interacts w/ 30s ribosomal unit
Initiation of translation
Initiation factors/GTP bind to 30s subunit
Initiator tRNA + mRNA join complex, large 50s subunit joins completing complex
ATP + GTP used to form ADP + GDP, IF3 dissociates
Elongation
P site where peptide bonds form , tRNA-met only tRNA that can bind to P site
Elongation factors needed -> ternary complex formed
A site (aminoacyl-tRNA) where incoming ternary complexes bind
Association of EF-G-GTP , ejection of empty tRNA from P site
Ribosome translocates, freeing up A site
Termination
RF-GTP binds to A site where termination codon appears
3 prokaryotic release factors
Hydrolysis of polypeptide chain from tRNA + dissociation of tRNA + RFs
tRNA structure + function
Anticodon, a. acid acceptor arm on 3’ end
Facilitated by aminoacyl-tRNA synthetases, specific for tRNA-amino acid complex (proofreading)
How can tRNAs recognise more than one codon?
Wobble - allows unconventional base pairing between 3rd base in codon and 1st base in anticodon
3’ end codon/ 5’ end anticodon loose base pairing
Inosine (altered guanine) can pair w/ A, C and U
Mutation
Alteration in nucleotide sequence of a DNA molecule (propagated as DNA replicates)
Errors in DNA replication
Causes spontaneous mutations
Arise due to base tautomerism - tautomers are isomers w/ slightly different chemical structures
keto-guanine -> enol-guanine
Mutagens
Base analogues - 5bU is analogue of thymine (still pairs w/ A), enol-5bU tautomer very common + pairs w/ G not A
Direct structural change - deaminating agents change nucleotide structure, deamination of adenine give hypoxanthine which pairs w/ C not T
e.g. deamination of cytosine gives uracil
e.g. deamination of guanine gives xanthine which blocks DNA replication
Other mutagenic agents that cause structural change
Alkylating agents - add alkyl group
Intercalating agents - inserts between base pairs
UV causes base dimerization - not easy to fix
Heat causes detachment of bases - creates AP site
Direct repair of mutations
Enzyme corrects nucleotide alteration
e.g ADA enzyme in E.coli can remove alkyl groups, MGMT enzyme in humans removes alkyl groups position 6 of G
e.g. Base dimers from UV can be repaired, DNA photolyase in E.coli
Excision repair
Damaged nucleotide removed + gap filled by DNA synthesis
2 types: base excision, nucleotide excision (longer piece of DNA)
Mismatch repair
Corrects errors in DNA replication, parents strand has correct nucleotide, daughter strand has mismatch
e.g. in E.coli mismatch in daughter strand recognised by MutH and MutS enzymes, MutH cuts DNA excising error
- less understood in humans + more complex
Non-homologous end joining
Corrects double strand DNA break, telomeres mark natural ends so can be distinguished from breaks
In humans, Ku proteins attach to broken ends + attract one another, DNA ligase joins ends together
How can you reverse the effect of a mutation?
In 2nd site reversion, 2nd mutation restores correct a.acid sequence despite nucleotide sequence still being altered
Monogenic disorder
Inherited diseases caused by defects in individual genes
6,000 known disorders e.g. cystic fibrosis
Effects of mutation on cystic fibrosis gene
Mutation in CFTR gene causes dysfunctional salt/water balance
Recessive disorder so needs mutation in both alleles to be affected
- F508del deletion of 3 nucleotides means CFTR protein still made but does not reach cell mem.
- G542X nonsense mutation changes glycine to uracil (stop codon created), CFTR protein not made + mRNA degraded
- G551D non-synonymous point mutation, G to A codes for aspartic acid, CFTR protein made + gets to cell mem. but only works at 4% of normal rate
Treatment for cystic fibrosis
Orkambi made from lvacaftor and lumacaftor
-> effective in people w/ 2 copies of F508del
- lumacaftor improve conformational stability of CFTR so can reach cell mem.
- llvacaftor is a CFTR potentiator which increases Cl- ion transport
Haploinsufficiency
When not enough gene product formed due to a mutation in just one allele, so chromosome directs synthesis through single functional allele
