eukaryotes - complexity Flashcards
if we take the dna from any of our cells and stretch it end to end what would its length be
2 metres
what does the way that dna is tightly folded make it available for
enzymes in the cell when it is needed
replication, dna repair, use its genes to make proteins
what is the first structure in the packing of dna
chromatin - BEADS ON A STRING
1 bead is made up of
- 146 base pairs
- histone proteins
how many histone proteins does each nucleosome contain
HISTONE OCTOMER
- 2 copies of each of the 4 types of histones
- found at the centre of the nucleosome
what are the 4 core histones
H2A
H2B
H3
H4
what are the properties of histones
- rich in Lysine (Lys, K) and Arginine (Arg, R)
- SO POSITIVELY charged at physiological pH
how do the properties of a histone protein enable it to bind to dna
- histones positively charged at pH 7
- DNA negatively charged (phosphate group)
- so bind to form a DNA histone complex
what is the name for a DNA histone complex
NUCLEOSOME
what does a nucleosome consist of
- little less than 2 turns of double stranded DNA
- DNA is wrapped around an octamer core of histone proteins (8 histone proteins)
what do repeating nucleosome units appear as under a microscope
“beads on a string” like structure
what is the next level of packing of dna following nucleosomes
CHROMATIN STRUCTURE
what does a chromatin consist of
- string of nucleosome condensed to helical array consisting of 6 nucleosomes per turn of helix
- generates a chromatin
what is the diameter of a chromatin
36nm
where are chromatin structures found in the cell cycle
- in interface chromatin
- in mitotic chromosome
which 2 lesser understood stages follow chromatin fibres in dna compaction
- further condensation of chromatin
- entire mitotic chromosome (metaphase)
what do the lesser understood levels of organisation seem to involve
- series of loops and coils
- these lead to thicker structures
how is the final packing seen in the metaphase chromosome achieved
further condensation of the chromosome when the cell enters mitosis during cell division
list the 5 stages of dna packing
1) dna double helix
2) nucleosome (dna wrapped round histone octomer)
3) chromatin fibre
4) further condensation of chromatin
5) entire mitotic chromosome (metaphase)
list the 4 stages of the cell cycle
1) G1 phase
2) S phase
3) G2 phase
4) M phase
what happens in the G1 phase
- cell receives signal to divide
- increase in size
- metabolic activities change
- to prepare cell for S phase
how does the cell ensure it is prepared for the S phase (synthesis) during the G1 stage
cell cycle control mechanism activates at G1 checkpoint
when does the cell move into the S phase
- once the restriction point has been passed during G1 phase
- cell committed to division
what happens in S phase
- SYNTHESIS phase
- dna replication
- produces 2 identical sets of genetic information (2 sets of chromosomes)
what happens in the G2 phase
- cell produces new proteins necessary for the M phase stage
what occurs again in the G2 phase which is also seen in G1
- cell cycle control mechanism at G2 checkpoint
- determines if cell can move to M phase
what happens during the M phase
- MITOSIS phase
- 2 identical daughter cells produced
what is the final stage of the M phase and what is it characterised by
- cytokinesis
- nuclear adhesion followed by cell division
what is dna replication
process of duplication of the entire genome prior to cell division
biological significance of dna replication:
why is extreme accuracy necessary
to reserve the integrity of the genome in successive generations
biological significance of dna replication:
what does the slower replication rate in eukaryotes result in
higher fidelity / accuracy of replication
what is the structure of dna in prokaryotic cells
- circular chromosome containing a single origin of replication
- generates 1 replication bubble with 2 replication forks
what does the structure of eukaryotic chromosomes suggest about their replication
- larger, more complex packing
- must be replicated from many points
- so have multiple origins of replication which initiate replication almost simultaneously
when dna does replication begin in eukaryotes
when initiator protein dna complexes are formed at specific dna sequences in the replication origins
how does the initiator protein dna complex start the process of replication
1) loads dna helicase onto the dna template
2) because they identify specific base on origin of replication
2) forming 2 replication forks at each origin of replication
how is the whole replicated strand formed
- forks extend in both directions as replication proceeds
- creates a replication bubble
- replication bubbles eventually all meet
how is the mechanism of replication in eukaryotes essentially the same as for prokaryotes
- replication starts once replication forks are established from the origin of replication
- both leading (continuous synthesis) and lagging strands (discontinuous)
- synthesised in 5’ to 3’ direction
- same machinery and proteins and enzymes
what is the role of dna polymerase in eukaryotic dna replication
adds nucleotides (complementary to template strand) one by one to the growing dna chain
how many dna polymerases are known in eukaryotes and which 5 have major roles during replication and have been well studied
14
- polymerase alpha
- polymerase beta
- polymerase gamma
- polymerase delta
- polymerase epsilon
what is the end problem of linear dna replication
- because dna linear
- problem with replicating the ends of the chromosomes (telomeres)
- dna polymerase can
1) only add nucleotides in 5’ to 3’ direction
2) cannot initiate synthesis of dna
3) can only extend dna with 3’ -OH available
in eukaryotic replication what happens in the leading strand
1) synthesis continues until the end of the chromosome is reached
2) so whole leading strand replicated
in eukaryotic replication what happens in the lagging strand
1) dna synthesised in a short stress
2) each of which is initiated by a separate primer
3) when replication fork reaches end of the linear chromosome
4) last primers are removed by 3’ to 5’ exonuclease activity of dna polymerase
5) there is NO way to replace the primer on the 5’ end
6) so dna at 5’ end of the chromosome remains unpaired
what happens to eukaryotic chromosomes over time due to the unpaired nature of the 5’ end of the lagging strand
the ends (TELOMERES) get progressively shorter and shorter as cells continue to divide so each round of replication generates shorter dna
why cant the gap left at the 5’ end of dna be filled by dna polymerase
no 3’ -OH available to which a nucleotide could be added
how is the shortening of eukaryotic dna prevented and what are these
TELOMERES
- protective cap at ends of each chromosome
- repeated dna sequence produced by telomerase
how do telomeres protect the dna against being shortened
- repetitive sequences that code for no particular genes
- protect important genes from being deleted as cells divide and strands shorten
what would happen if we didnt have telomeres
- nucelases would digest the single stranded 3’ overhangs
- daughter dna become shorter than parent dna
- eventually untied dna lost
what is telomere shortening related to
- replicative potential of cells
- cell lifespan
- when telomere length approaches a critical level, cells begin ageing, stop dividing and die
some cells express which enzyme that reverses a telomere shortening and extends the telomeres
telomerase
what is telomerase
- ribonucleic protein enzyme
- consists of an enzyme + built in RNA molecule
- rna dependent polymerase
how does telomerase extend the 3’ ends to allow extension of the telomere
- contains an rna region that it uses as a template to make dna and extend the end
what are the 3 steps undertaken by telomerase to finish the replication of linear chromosomes
1) BINDING
telomerase binds to rna molecule containing a sequence complementary to telomeric repeat
2) POLYMERISATION
extend 3’ end of telomere adding nucleotides to overhanging strand using complementary rna as a template
overhanging strand of the telomere dna then elongated following repeated extension of 3’ end of lagging strand
3) TRANSLOCATION
one 3’ end of lagging strand template sufficiently elongated, 5’ end is extended by normal lagging strand synthesis
how often does the binding polymerization-translocation cycle of telomerase occur
many times
telomere formation: explain step 1: binding
telomerase binds to rna molecule containing a sequence complementary to telomeric repeat
telomere formation: explain step 2: polymerisation
- extend 3’ end of telomere adding nucleotides to overhanging strand using complementary rna as a template
- overhanging strand of the telomere dna then elongated following repeated extension of 3’ end of lagging strand
telomere formation: explain step 3: translocation
- once 3’ end of lagging strand template sufficiently elongated
- 5’ end is extended by normal lagging strand synthesis
where does telomerase operate
ONLY IN SPECIAL CIRCUMSTANCES
- cells that divide rapidly (germ, foetal, stem)
- cancer cells
- role in cellular aging
how does telomerase enable cancer cells to continue dividing uncontrollably
- they contain actin telomerase
- enables them to become “immortal” cells
where doesnt telomerase operate
- adult cells
- silent in vast majority of human tissues
what are the differences between prokaryote and eukaryote replication
origin of replication
- p = single
- e = multiple
rate of replication
- p = 1000 nucleotides/s
- e = 50 - 100 nucleotides/s
dna polymerase types
- p = 5
- e = 14
telomerase
- p = not present
- e = present
rna primer removal
- p = DNA polymerase 1
- e = RNase H
strand elongation
- p = DNA polymerase 3
- e = pol alpha, pol delta + pol epsilon
what does the process of gene expression in eukaryotes enable protein synthesis
process by which the genetic code of a gene is used to direct protein synthesis
what are genes that code for amino acid sequences called
structural genes
what are the main stages of gene expression
transcription
translation
which strand of dna does transcription use (of the 2 exposed strands in the transcription bubble)
- template strand (makes an mrna strand complementary to this strand)
(the other strand = coding strand)
what does the coding strand contain
same sequence as the mRNA but thymine in the place of uracil
where does transcription occur in eukaryotic cells and what must occur before the transcription product is transported to the cytosol for translation into a protein
NUCLEUS
- mRNA precursors processed
which enzymes perform transcription in eukaryotes
1) rna polymerase 1
- transcribes 5.8S, 18S, 28S rRNA genes
2) rna polymerase 2
- transcribes all proteins coding genes
- genes for regulatory rnas (sno, mi, si and most sn RNA)
3) rna polymerase 3
- transcribe structural rnas (5s rrna, trna, sn rna)
what are rRNAs named according to, what does this mean
- “S” values
- larger value = larger rRNA
what 4 sequential stages are involved in eukaryotic transcription
1) initiation
2) elongation
3 termination
4) processing
transcription: what happens in stage 1 initiation
- dna molecules unwind and separate
- forms small open complex in promotor region
- rna polymerase binds to promotor of template strand
transcription: what happens in stage 2 elongation
- rna polymerase moves along dna template strand 3’ to 5’ adding new nucleotides
- synthesising an mrna molecule in the 5’ to 3’ direction
transcription: what happens in stage 3 termination
transcription terminates at a specific region
transcription: what happens in stage 4 processing
- eukaryotic rna transcripts = immature after transcription (pre-mRNA)
- rna molecules processed in nucleus before being transported to the cytosol
- introns removed and exons spliced together
transcription initiation:
what are promoter regions
- where rna polymerase and helper proteins bind to dna of genes to initiate transcription
- regions immediately upstream of the transcription start site
transcription initiation:
what nucleotide sequence do many eukaryotic promoters have
- TATA BOX
- highly conserved
- between -30 and -25
transcription initiation:
how does the TATA box enable creation of the transcription bubble
- recognised by 1 transcription factor
- allowing other transcription factors + eventually rna polymerase to bind
- contains many adenines and thymines SO easy to pull strands apart
transcription initiation:
what other eukaryotic promoter recognition sequences have been recognised
CAAT BOX
- found at -75 base pairs
- consensus sequence GGNCAATCT
- N = thymine or cytosine (depends on gene)
- determines efficacy of transcription
GC BOX
- GGGCGG
transcription initiation:
how can rna polymerase bind to a dna template
1) transcription factor binds to promoter region
2) then recruit appropriate polymerase
3) completed assembly of transcription factors and rna polymerase bind to the promotor forming transcription pre-initiation complex (PIC)
transcription initiation:
how does rna attach to the promoter in eukaryotic cells
- not directly
- rna polymerase II requires many transcription factor proteins (activator, mediator, chromatin modifying proteins)
transcription initiation:
why does eukaryotic dna require transcription factors when prokaryotic does not
- nucleosomes and higher order DNA structures
- advanced regulatory mechanisms
transcription initiation:
in what ways is gene expression regulated
1) chromatin remodelling
2) enhancers + silencers
3) modification
transcription initiation:
what is chromatin remodelling to control gene expression
- chromatin rearranged
- from condensed to transcriptionally accessible state
- allows transcription factors / dna binding proteins to access dna to control
transcription initiation:
what are enhancers and silencers to control gene expression
enhancers = activators (transcription factors) bind to enhancer region, activate transcription/inc rate
silencers = repress transcription/ slow rate when bound to repressors
transcription initiation:
what properties do enhancer regions have
- 1000s nucleotides
- from coding (exon) OR intron seq
- some are conditional (only work in presence of other factors + transcription factors)
transcription initiation:
what is modification to control gene expression
- methylation occurs at cytosine bases of eukaryotic dna
- common epigenetic signalling tool cells use to lock genes in off position
- blocks promoters for activator transcription factors = cannot bind
transcription termination:
what is the signal for rna polymerase to stop transcribing
polymerase transcribes a terminator (signal of dna)
transcription termination:
how is rna polymerase II stopped
- 1000-2000 nucleotides beyond end of gene being transcribed
- this is a pre-mrna tail + is removed by cleavase during processing
transcription termination:
how are rna polymerase I + III stopped
- require termination signal
- genes they transcribe contain a specific 18 nucleotide sequence recognised by termination protein
- involves mrna hairpin
transcription processing:
what 3 types of processing does pre-mRNA go through
1) addition of the 5’-cap
2) addition of a poly A tail
3) splicing
transcription processing:
what is addition of the 5’-cap
- 7-methylguanosine with 5’ to 5’ phosphate bonds
- added to 5’ terminus of mRNA (cup structure)
transcription processing:
why is the 5’ cup essential when mRNA is used for protein synthesis
binds to ribosomes via special proteins attached to this cup
transcription processing:
what other roles does the 5’ cup have
- protects rna from degradation by ribonucleases
- helps ribosome attach to mrna during translation to make proteins
- role in transporting mature mrna out of nucleus
transcription processing:
what is addition of a poly A tail
- pre-mrna cleaved by endonuclease containing protein complex between AAUAAA and GU rich sequence
- poly(a) polymerase adds 100-1000 adenosine nucleotides to 3’ end of just cleaved pre-mrna (forming poly(a) tail)
transcription processing:
why is addition of a poly A tail important
- initiation of protein synthesis
- inhibition of mrna degradation
transcription processing:
what is gene splicing
- removes introns
- ## joins exons together
transcription processing:
which eukaroytic genes do not contain introns
interferons
transcription processing:
what is the structure of an intron
- 5’ end = 5’ splice site normally signified by GU as first nucleotides
- branch site = region w highly conserved adenosine downstream
- 3’ end = AG
what is the 2 step splicing mechanism used for mRNA
STEP 1
- 2’ OH of branched site attack phosphate at 5’ of spliced site
- generates lariat intermediate
- release exon 1
STEP 2
- 3’ OH in exon 1 attack phosphate at 3’ end of exon 2 splice site
- then ligate exon 1+2 together
- lariat form of intron released
which enzyme catalyses splicing
spliceosome (large ribonucleic protein complex)
what is the structure of a spliceosome
1) U1 snRNA base pair the 5’ splice sites
2) U2 snRNA base pairs w the branch site
3) U1 joins U2 form a complex before being joined by other subunits U4, U5, U6
4) undergoes series of rearrangements to catalyse splicing
what is alternative splicing
- select diff combos of splice sites in mrna precursor to produce variably spliced mrnas which encode proteins that vary in their sequence + activity
- several forms of a protein (isoforms) produced from same gene
where does alternative splicing have significant roles
- gene expression
- protein diversity
- 95%
what % of multi exon genes undergo alternative splicing
95%
where does alternative splicing occur relative to dentistry
in production of multiple forms of amelogenin (tooth enamel protein)
