Nucleic acids, Bacteria and organelles Flashcards
What did Frederick Griffiths experiment with a non virulent and virulent strains of streptococcus pneumoniae show?
Showed that a non virulent strain could be converted to a virulent strain by mixing it with a heat killed virulent strain
Showed something was being transferred but hadnt yet identified what
What did Oswald and Avery’s experiments show?
Extracted DNA, RNA, Carbohydrate, protein and lipid from heat killed virulent strains and mixed each with the non virulent strain, only the DNA converted it to a virulen strain
Showed DNA was responsible for the transformation
What is Hershey and Chase’s experiments with bacteriophages show?
Bacteriophage T2 = virus made up of DNA/RNA core and a protein capsule
Grew one in radioactive sulphur - incorporated into proteins
Grew another in radioactive phosphorus - incorporated into the DNA
Only when bacteria were infected with bacteriophage grown in the radioactive phosphorus could radioactivity be seen
Confirmed Oswald and Avery’s findings
What did Watson and Crick’s discover and who’s work did they use to help?
Discovered the double helix formation of DNA
Used Rosalind Franklins X-ray crystallography
Used Wilkin’s evidence that %A=%T and %C=%G
They also recognised that this pointed to a mechanism for DNA replication
What is the sugar in DNA?
Deoxyribose
How are adjacent nucleotides joined in DNA?
Adjacent dexoyribose sugars are joined by phosphodiester bonds
How many fused rings of nitrogen do the purine bases contain and which are they?
They contain 2 fused nitrogen rings
Adenine and Guanine
How many fused rings of nitrogen do the pyridamine bases contain and which are they?
They contain 1 fused nitrogen ring
Cytosine, Thymine, Uracil
How are the bases in DNA joined?
H bonds
How many H bonds join A and T?
2
How many H bonds join C and G?
3
How is DNA arranged in eukaryotes compared to prokaryotes?
Coiled into chromosomes in eukaryotes (wound round proteins called histones)
Dispersed in cytoplasm in prokaryotes (no membrane bound organelles so no nucleus)
Is RNA double or single stranded?
Single
Which base is changed in RNA compared to DNA?
Uracil replaces thymine in RNA
What sugar makes RNA?
Ribose
How can the secondary structure of RNA be formed?
Base pairing between complementary bases of the same strand
Why is DNA replicationconsidered ‘semi conservative’?
Because the daughter DNA strands contain one strand of the original DNA and one newly synthesised strand
Which experiment proved the semi conservative nature of DNA replication and how briefly did it work?
Meselson Stahl experiment
Bacteria grown in heavy nitrogen (N15)
Then grown in light nitrogen (N14)
After rounds of replication, centrifuged, heavy = lower down in centrifuge
What is the ‘replication fork’?
Site at which DNA unwinds so DNA replication can begin
What is the origin of replication and what is there significance in eukaryotes?
Replication fork forms at the origins of replication
Have many of these in eukaryotes to speed up replication
Which enzyme is involved in unwinding DNA and seperating the DNA strands?
DNA Helicase
In DNA replication what holds the strands apart?
Single stranded binding proteins
What enzyme carries out DNA synthesis in DNA replication?
DNA polymerase
One on each strand, carry out DNA synthesis
Which direction does DNA replicate?
5’ to 3’
What are RNA primers, which enzyme in involved in their synthesis and why are they needed in DNA replication?
Short single stranded sections of RNA
Synthesised by DNA primase
Need a primer as need a free -OH group for the DNA polymerase to attach nucleotides to
What enzyme catalyses the formation of phosphodiester bond between 5’ phosphate group and 3’OH group?
DNA polymerase III
What are dNTPs and what is their role in DNA synthesis?
deoxynucleoside tri phosphates
Incoming dNTPs, bond forms between -OH group on 3’ end of strand being synthesised and phosphate group on dNTPs and then the other 2 phosphate groups from the dNTPs are released as pyrophosphates
This is a source of energy
Which is the leading strand and which is the lagging strand in DNA replication?
Leading strand is the 3’ to 5’ template
Lagging strand is the 5’ to 3’ template
In what pattern is DNA synthesised on leading strand template?
Contiuously
In what pattern is DNA synthesised on lagging strand template?
As okazaki fragments
DNA synthesis is in the opposite direction to the movement of the replication fork
DNA primase is required to lay down primers at regular intervals
Once RNA primers have been removed which enzyme is involved in replacing the RNA primers with DNA and which enzyme is involved in sealing the gaps?
DNA polymerase 1
DNA ligase seals the gaps
What is the difference between a nucleotide and a nucleoside?
Nucleotide = base + sugar + phosphate group Nucleoside = base + sugar
What are the 2 checking mechanisms we have for minimising error in DNA replication?
1) DNA polymerase has a 3’ proof reading function
2) Mismatch repair enzymes
EMS (ethyl methone sulphonate) is an example of a chemical carcinogen, how does it work?
EMS reacts with DNA to form 06-ethylguanine adduct which mispairs with thymine rather than cytosine at replication
Get G-C to A-T mutation
What are the 4 types of mutation?
1) Deletion
2) Substitution
3) Insertion
4) Rearrangement
What is the difference between base excision repair proteins and nucleotide excision repair proteins?
Base excision repair proteins cute out damaged bases
Nucleotide excision repair proteins are less specific and cut out whole sections of DNA
What does the enzyme Uracil-N-glycosylase do, in terms of DNA repair?
Uracil-N-glycosylase recognises the presence of uracil and cuts it out
Uracil can arise in DNA due to the deamination of cysteine
DNA polymerase I replaces the DNA and DNA ligase seals the gap
What are the 3 shapes of bacteria?
1) Spiral - Spirilla
2) Rod-shaped - Bacillus
3) Round - Cocci
What is the rough structure or prokaryotes?
Dispersed cytoplasm No membrane bound organells Inner membrane Rigid outer cell wall Outer membrane (but only in Gram negative bacteria)
What is the process of GRAM staining?
1) Fixation
2) Crystal Violet (goes blue)
3) Iodine (goes purple)
4) Decolourization
5) Counter stain (safranin - red)
What happens to Gram negative and Gram positive bacteria when counter stain is added?
Gram negative - do counter stain = RED
Gram positive - dont counter stain = PURPLE
What are bacterial cell walls made up of?
Peptidoglycan
Which type of bacteria contains lipoteichoic acid in its cell wall?
Gram positive
Which type of bacteria has a 2nd outer membrane?
Gram negative
Why do gram positive bacteria not counter stain?
Adding acetone or alcohol (Decolourisation) reacts with the lipid membranes and removes the outer layer to expose the peptidoglycan layer
In gram negatives this is thin so the stain is washed away and they counter stain
In gram positives this is multilayered so the crystal violet-iodine complexes are retained and they dont counter stain but stay blue
What is the function of a cell wall in bacteria?
1) Protects from lysis in hypotonic solutions
2) Protects from physical damage
3) Controls access of some chemicals to cell membranes
What are endospores?
Tough spherical forms that bacteria can assume that resist extreme temps,
Bacteria assume these in response to adverse environments and can remain dormant for thousands of years
Where is the capsule in gram negative and gram positive bacteria and why can this make gram negative more resistant to host cell phagocytosis?
Gram positive capsule is outside cell wall
Gram negative capsule is inside outer membrane, antigenic proteins are located on the capsule and in gram negative these antigenic proteins can be hidden making them resistant to host cell phagocytosis
What are pili (fimbriae) and what do they allow bacteria to do?
Long, stiff structures which enable bacteria to stick to the cells of the host through specialised molecules called adhesins
Although pili are immunogenic they can change their antigens so they can avoid immune recognition
What is the genome of an organism?
Entire genetic content
What was the purpose of the Human Genome Project?
Project set up to sequence human genome
Results tell us there are about 20,000 genes that code for proteins but non coding sequences could code for mRNA to control gene activity eg. controls what are turned on and off
What method was used to sequence the human genome?
Sanger dideoxy sequencing
What is required in the medium for sanger dideoxy sequencing?
1) 4 kinds of dNTPs
2) RNA primers
3) DNA polymerase III
4) 1 kind of ddNTP
What is the difference between a dNTP and a ddNTP and what does this mean for DNA synthesis?
ddNTPs have no 3’OH group so no more dNTPs can be added
What process is used to separate out the strands of DNA is Sanger dedioxy sequencing?
Gel electrophoresis
Used to radioactively label and look at the strands
Now fluoroescently label and computer reads, much quicker, more DNA can be processed
What is a genetic polymorphism?
Specific mutation that occurs in more than 1% of the population
What are single nucleotide polymorphisms?
Polymorphisms (occur in more than 1% of the population) where only one nucleotide is changed
There are links between single nucleotide polymorphisms in non coding sections of DNA and disease
What is the difference between genotype and phenotype?
Genotype = actual DNA sequence Phenotype = physical presentation
What are epigenetic markers?
Dont cause a change to the actual DNA sequence but can be modifications to the chromatin or histone (methylation, phosphorylation, acylation (including DNA methylation)) which modify which genes are expressed
For example genes can be silenced by epigenetics
Can epigenetic markers be inherited?
Yes is stable
How can epigenetic markers be reversed?
May be reversed by environmental influences eg. diet or exposure to carcinogens
DNA methylation is a form of epigenetic marking which is used to silence genes, what sites can methylation occur at, what enzyme catalyses it and what is the methyl donor?
Methyl groups added to cytosine at CpG sites
DNMT (DNA methyltransferase) is the enzyme
SAM is the methyl donor
If DNMT misses a cytosine at CpG it may result in a gene being expressed where it would otherwise be silenced
How has the human genome project helped medicine?
1) Improved understanding of molecular pathology of disease
2) Predicting risk of disease
3) Improved diagnosis
4) More effective drugs: targeted, tailored with less adverse reactions
How many bases in the human genome?
6 million
What percentage of 2 different organisms genomes are identical?
99.6%`
What is a DNA microarray?
Systematic way to survey DNA and RNA variation
Place thousands of fragments of DNA onto a chip , fragments of DNA added then bind to complementary strands, the presence of a label at that site tells you that binding has occurred and that the DNA contains that sequence
What are the uses of DNA microarrays?
1) Compare polymorphisms between different individuals
2) Compare gene expression between normal and diseased tissue
3) Compare gene expression between different types of cell
What are the units from which RNA is synthesised called?
Ribonucleotides
What is the template strand and what is another name for it?
Strand from which RNA is transcribed, contains the genetic information
Also called the anti sense strand as the mRNA transcribed from it is an exact copy of the non transcribed ‘sense strand’
What enzyme is needed for transcription and how does it differ from DNA polymerase?
RNA polymerase
Differs as it does not require a primer
What is the substrate of RNA polymeras?
ribonucleoside triphosphates
During transcription, mRNA does not remain bond to the DNA but detaches behind the RNA polymerase, what does this ensure?
More than one strand of mRNA can be synthesised along one strand of DNA at any one time
Prokaryotic cells only have one type of RNA polymerase, how many types do eukaryotes have and what do they synthesise?
3
1 and 3 synthesise tRNA and rRNA
2 synthesis mRNA
What are promoter regions of genes and what sequence do they contain?
Initiator sites for transcription
Contain the TATA box which is located 25 nucleotides from transcription is initiated
What happens at the TATA box and why?
Box to which transcription factors bind which initiate transcription
Weaker links between A and T (2 H bonds not 3) make it easier to unwind DNA at this region
General transcription factors are needed for transcription, what is their role?
1) Position RNA polymerase at the promoter region
2) Hold DNA strands apart
3) Release RNA polymerase from the promoter region to go into elongation mode
How is transcription stopped?
When the RNA polymerase reaches a terminator sequence
Produce secondary structures of RNA which are unstable and cause the RNA polymerase to release the chain
Where does splicing occur?
In spliceosomes in the nucleus (containing small nuclear RNAs which process pre-mRNA using energy)
Doesnt occur in eukaryotes as they have no introns
What happens during splicing?
Introns are removed so only exons remain
Other than splicing what other 2 processes occur to modify the pre-mRNA?
1) Capping
2) Cleaving
What is Capping or mRNA?
the 5’ end of mRNA is capped by a 7methyl guanine
Carried out by 3 enzymes
Capping signifies the 5’ end of the mRNA and distinguishes it from other types of RNA
What is cleaving of mRNA?
Once termination sequence is reached
The 3’ end of mRNA interacts with RNA binding and RNA processing proteins
3’ end is cleaved and a poly A tail is added (200 adenine nucleotides)
Poly A binding proteins bind here and help guide the synthesis of protein at the ribosome
What are the 3 stop codons?
UAA
UAG
UGA
Which end of the tRNA molecule is the amino acid bound to?
The 3’end
What loop of tRNA is the anticodon located on?
The 2nd loop
How many nucleotides is tRNA made up of?
around 80
What is wobble base pairing?
Ability of a tRNA molecule to bind to a codon that differs at the third nucleotide
Once tRNA has been synthesised which enzyme is involved in ‘charging the tRNA’ with its amino acid?
aminoacyl-tRNA-synthetase, each amino acid has a specific aminoacyl-tRNA-synthetase which attaches it to its appropriate tRNA molecule
How many subunits make up a ribosome?
2
What codon does translation begin at?
AUG
a special tRNA is required to initiate translation
This initiator tRNA always carries methionine
What amino acid is always located at the N terminal of a newly synthesised protein?
Methionine
But this is normally removed by proteases
What binds to the ribosome along with the initiator tRNA and how do they recognise initiator tRNA?
Eukaryotic initiation factors bind along with intiator tRNA
The recognise tRNA as it has a different codon to the tRNA which normally carries Met
What do the large sub unit and small subunits of the ribosome bind to initially?
1) Large subunit binds tRNA and eukaryotic initiation factors
2) Small sub unit binds to mRNA
What is a polysome?
Structure formed when small and large sub unit of ribosome come together on the mRNA
What process occurs to begin translation?
1) Small sub unit moves along mRNA until it finds AUG then translation is initiated
2) EIF’s and other initiation factors dissociate and large subunit (also bound to tRNA) binds
What are the 3 sites on the large subunit of a ribosome and what happens at each?
EPA
E - where tRNA exits from the ribosome - gets ‘uncharged’
P - already bound to tRNA molecule
A - site of newly binding tRNA molecule
tRNA molecules bought into close proximity
What catalyses the formation of peptide bonds between amino acids?
Ribosomes
peptidyl tRNA forms peptide bond
What happens to terminate translation?
When the ribosome reaches a stop codon, translation is terminated
Release factors are involved in this
After translation polypeptides are modified and this determines the final structure of a protein
How do Abx inhibit bacterial protein synthesis and why doesnt it affect body cells?
Abx lodge in pockets of ribosomal RNA
As processes are slightly different in prokaryotes and eukaryotes, such processes dont affect body cells
Tetracycline and Streptomycin bind in the small subunit
What are the 2 methods by which membrane proteins can be synthesised?
1) Proteins with a specific amino acid terminal sequence are translocated to the ER
2) Proteins are made in the cytosol and anchored to membrane after synthesis is complete
How are membrane proteins that are translocated to the ER synthesised?
1) Some of protein gets translocated to the lumen of the ER
2) Sequence of about 20 hydrophobic amino acids gets incorporated into the bilayer
3) Signal sequence gets cleaved off inside the ER
4) Rest of protein gets synthesised in the cytosol and doesnt go into the ER lumen (unless there is an additional amino acid sequence to cause it to be translocated again)
How are membrane proteins that are made in the cytosol anchored to the membrane?
1) Particular carboxyl terminal sequence causes them to be synthesised like this
2) Cys residue at the N terminal is linked to a fatty acid chain in the membrane
3) Carboxyl terminal is cleaved
What is the difference between euchromatin and heterochromatin?
Euchromatin is diffuse and actively transcribed
Heterochromatin is condensed and is inactive
What is the nucleolus?
Area where the ribosomes are assembled
What is nucleoplasm?
Substance in the nucleus which surrounds chromatin
What is the nuclear envelope?
Double layered membrane with pores
Inner membrane is smooth
Outer membrane is continuous with the ER and covered in ribosomes
What are cistae?
Flattened sacs of the ER and the golgi apparatus
What is the function of the SER?
Manufactures phospholipids, cholesterol, steroid hormones
Enzymes in the SER involved in detoxification of lipid soluble molecules transforming them into polar metabollites which can be excreted by the kidneys
What is the large subunit and small subunit of the ribosome made up of?
Large subunit - 1 long strand and 2 short strands of RNA + 50 proteins
Small subunit - 1 long length of RNA and 33 proteins
What is the function of the free ribosomes?
Just make proteins for the cytoplasm and nucleus
What codes for the ribosomal RNA of the ribosomes in the RNA?
Mitochondrial DNA
What is the function of the golgi apparatus?
Concentration and modification of proteins produced in the ER
Moved by being shuttled from cisternae to cisternae in vesicles
What is the function of lysosomes?
Other membrane bound organelles fuse with them so contents can be digested eg. bacteria and viruses
Used to destroy cell organelles which are first engulphed by the ER
What is a proteosome?
Large protein complexes - 4 rings around a core
Proteins destined for destruction are tagged with ubiquitin
What is a peroxisome?
Small vesicles which resemble lysosomes but contain different enzymes (oxidases and catalases)
Oxidases break down a variety of substrates eg. fatty acids with the production of H2O2
Catalases use H2O2 to oxidise other toxic compounds or break down H2O2 to water
Other peroxisome enzymes are involved in synthesis of cholesterol and other membrane components
What are the 3 types of protein fibres which make up cytoskeleton?
1) Microfilaments
2) Intermediate filaments
3) Microtubules
What is the function of microfilaments in the cytoskeleton?
Twisted double strands of Actin
Enables cells to move and change shape, form a mesh below the plasma membrane to which proteins are attached
Enables cell to fix to its external environment and also form core of microvilli
What is the function of intermediate filaments in the cytoskeleton?
Very stable, give mechanical support to nucleus and plasma membrane
alpha helical dimers
What is the function of microtubules in the cytoskeleton?
Can be lengthened and shortened
Used as tracks along which organelles and proteins can be moved to specific locations
What kind of cytoskeletal proteins are flagella and cilia formed from?
Microtubules
