molecular biology (exam prep) Flashcards
basics
What are the characteristics of life?
-maintain integrity (boundaries)
-store information
-perform and regulate metabolism (energy)
-interact with other cells, signals and environments
-replicate/divide
Life: C-based and DNA-based
What are the life domains?
bacteria
archaea
eukarya
viruses
Comparison of bacterial and eucaryotic genomes
bacteria:
-are circular
-no teleomeres
-on the cytoplasm
-contain plasmids
-wraps around HU proteins
-located within an operon
-no mrna post transcirptional mods
eucaryotic:
-resides in the nucleus
-linear
-larger genome
-usually no plasmids
brewers yeast has plasmids
both
-has mitocondiral chloroplasts
origin of present day mitocondria
Endosymbiosis: bacterial cell engulfed by eukaryotic cell and evolve together.
comparison of genome sizes
the complexity of the organism doesnt always comprehend with the size of the genome
3 types of staining to visualise
-binding a molecule to a specific organelle structure
-binding an antibody
-GFP staining (green fluoresent)
modularity
an ordered assembly of amino acids that have already formed from atoms. Modularity allows evolution to occur by forming components that can be individually modified.
polymers
nucleic acids
proteins
lipids
polysac
monomers
nucleotides
amino acids
fatty acids
macromolecules
carbohydrates
lipids
protein
nucleic acids
Draw a structure of nucleic acids
(DNA, RNA)
nucleoside
example uridine
nitrogenous base and 5 carbon sugar
Similarites and differences DNA and RNA
similarities
DNA:
-base T
-double-stranded
-relatively stable
-information storage
-usually one
-deoxyribose sugar
RNA:
-single-stranded
-unstable
-base U
-many functions eg transport, enzymatic etc
the hypothesis the RNA proceeded DNA
DNA replication
semi conservative
According to the semiconservative model, after one round of replication, every new DNA double helix would be a hybrid that consisted of one strand of old DNA bound to one strand of newly synthesized DNA.
Then, during the second round of replication, the hybrids would separate, and each strand would pair with a newly synthesized strand. Afterward, only half of the new DNA double helices would be hybrids; the other half would be completely new.
okazaki fragments?
the short lengths of DNA that are produced by the discontinuous replication of the lagging strand.
dna replication in bacteria
what occurs during initiation of protein synthesis?
the initiation of DNA replication takes place at the origin (ori c) in E.coli.
begins negatively supercoiled
9mer and 13 mer are a+t rich regions, thus less hydrogen bonds. (melt at lower temps and have less hydrogen bonds compared to G C)
10 or 20 monomers of DNAa (inihiator protein)
binds to 9 mer regions. 9 mer region wraps around DNA a monomer this induces the A+t rich region to unwind. (open complex)
DNAC helicase loader loads DNA b to begin unwinding DNA.
dna is repilcated at the repilcation fork, is bidirectional and semi discontinous
primosome
a protein complex responsible for creating RNA primers on single stranded DNA during DNA replication
7 proteins:
DnaG primase, DnaB helicase, DnaC helicase assistant, DnaT, PriA, Pri B, and PriC
how can primosomes be monitored in vitro?
using fluorophores, a fluoresent chemical that can remit light upon light excitation
technqiue: smfret
main initation proteins in e.coli
DnaA (initiator), DnaB (helicase), DnaC (loader), DnaG (primase)
DNA Pol III Holoenzyme synthesises both strands (5’→3’)
*DNA Pol I replaces RNA primers on lagging strand with DNA
*DNA ligase fills the gaps
topoisomerase in e.coli
catalyzes the relaxation of negatively supercoiled DNA
purines
draw structure
adenine
guanine
purimidines
thymine
cytosine
uracil
differences between dna replication in eukarya vs bacteria
What occurs in a reverse transcriptase reaction?
Reverse transcription involves the synthesis of DNA from RNA by using an RNA-dependent DNA polymerase.
The DNA strand is not identical to the og
function of teleomeres
bacteria with teleomeres
Linear chromosomes => telomeres *short DNA repeats (e.g. TTAGGG) *G-rich strand + C-rich strand *every round of replication loses up to 200bp
Streptomyces
milestones:
Be more familiar with some important milestones in the development of molecular biology
Be able to discuss the Lac operon and how that relates to our understanding of genetic regulation
Recognise how molecular biology underpins biotechnology and can contribute to medicine, agriculture and basic science
Recognise links to fields such as gene editing and synthetic biology, which have grown out of molecular biology
Name 3 important milestones in molecular biology
-“jumping genes”
-lac operon
-pcr
what did barbra mcclintock discover?
the discovery of transposable elements in maize “the jumping genes” .
known as the AC/DS system, chromosome breakage occurs at the dissociation and is regulated by the activator which can also provide its own tranposition.
thus replication is not always linear
and the disruption caused by them on chromosome 9
what is the lac operon?
it is a group of genes that with a single promotor that encodes genes for the transport and metabolism of lactose in bacteria (e.coli)
what is the basic process of a functioning lac operon?
sugar production: allosterically repressed.
transcription only occurs when there is lactose ready to be digested
a repressor blocks the production of the
the operon contains 3 types of lactose enzymes (lac C, lac Y, Lac Z) which but if IPTG promotor is added it will bind to the repressor and allow the production to occur
cis elements that bind transregulators
binding sites for proteins:
promotor
operator
cbs-cap binding site
a major trans regulator encoded by the operon is LACI this is the repressor that binds to stops the polyerase binding to the repressor
e.coli’s lac z as a reporter gene
when cleaved by the β-galactosidase enzyme it produces blue product.
thus denoting if a gene is expressed or not
other reporter genes
firefly luciferase gene in plant cells and transgenic plants
reading genes
sanger
writing genes
chorana
alpha helix
– 3.6 amino acyl residues per turn; 2.3 Å helix radius.
*Most common helix in proteins.
* Usually about 10 aa residues, but can be 4-40+.
* Usually contains M, A, L, E, K aas but P and G disrupt a helix.
NAD+ Acidithiobacillus thiooxidans
310 helix
3.0 amino acyl residues per turn;
1.9 Å helix radius *
. * Very strained structure.
* Found in e.g. myoglobin and hemoglobin. * Usually very short - <4 aa residues.
blue whale myoglobin
pi helix
4.4 amino acyl residues per turn; 4.4 Å helix radius.
* Energetically unfavourable – selected against unless functionally critical, so found near active-sites.
* Usually seen as a bulge on a long alpha helix.
* Usually short – 7-10 aa residues, usually.
methane oxidising bacteria
soluble methane monoxygenase (13)
1 strand beta helices
right handed (one arrow)
Thermal hysteresis protein YL-1 aka antifreeze protein)
from mealworm
inhibit the formation of large ice grains inside the cells that may damage cellular organelles or cause cell death
freeze tolerance and ice adhesion
first discovered 50 years ago antartic fish present in millimolar concentrations their fucntion was to stop ice crystal growth
2 strand beta helices
Ice-binding protein (IBP) from a type of Flavobacterium
very potent for a microorganism produces FH in the range of 3°C submillimolar conditions
3 strand beta helices
Antifreeze protein isoform 501 from
spruce budworm.
this anti freeze protein protects organisms from freezing by adhering to ice crystals thus preventing their growth. they absorb to ice adhere to its morphology and prevent further growth.
theories suggest the effectiveness of this protein is due to how well it can stop freezing at the basal plane.
A study was conducted in 2008
protein structure and function part 2
tertiary structures :
other interactions occuring within the polypeptide chain.
predicting helices that span membranes
uses tericary structure of alpha helices grouped together
hydrophobicity plots such as kyte-doolittle plot allows us to examine a primary structure regions are.
graph: above 0 hydrophillic below 0 hydrophobic
example of this: aquaporin (lets what in and out of a cell). 7 alpha helices per sub unit in homosapiens.
beta propeller
multiple 4-stranded beta-meanders motifs arranged in the form of blades. all joined together.
active site of many enzymes in the centre of propeller.
example:
methanol dehydrogenases (enzyme)
8 bladed propeller common in alcohol dehydrogenases.
takes electrons off of the alcohol and donates them to cytochrome c. Thus couples directly to respiration.
from paracococcus bacteria.
beta barrels
(hydrophobic outside)
beta strands with a turn, anti parallel.
example: sensory protein
FhaA-receptor protein from e-coli
needs to be turned on for hole to open. (outside, detergent molecules show this )
2 types:
-outside hydrophobic (will disolve in a membrane)
-inside hydrophobic
beta barrels
(hydrophobic inside)
found in the cytoplasm.
Will dissolve in water (hydrophillic outside)
benzene
alpha solenoid
(bike chain)
stacked pairs of alpha helices
form large flexible structures found in things that need protein-protein interactions. Massive bendy surface area.
example:
phosphatase 2a
adds or takes phospate to or from proteins
quaternary structure (2types)
formed by polypeptide interactions
binding of co-factors ( non-protein chemical compound or metallic ion that is required for an enzyme’s role as a catalyst)
information flow
-operons (transcribe as on mRNA strand)
example in soluble methane monooxygenase.
redox co-factors
2 types
soluble (not part of proteins)
redox active enzymes: PQQ (quinone proteins, hydrophobic)
hemes
flavins
porphyrin rings
chlorophylls
cobalamins (b12)
bound metals
hemes
Transcription in bacteria & eukarya
Rna and sigma factor come together- high ifinity for DNA sequence.
Locate the promotor (strong association) forms closed promotor.
DNA strands start opening up, transcription starts for RNA.
Signals tell polymerase to stop
how does rna polymerase recognise where to start?
promotors: they are recongisable by 2 main sequences
one at -10 & -35 upstream from start of transcription
+1 start of transcription (purine normally)
-10 consesus often TATAAT
-35 consensus often TTGACA
distance most important
Top strand is coding strand
other is template strand therefore the outcome RNA will be the compliment of the top strand
synthesis always happens 5’- 3
differences in transcription
bacteria vs eukarya
e- 3 types of RNA polymerase
1- rRNA transcribe
2-mRNA transcribe
3-tRNA transcribe
more complex promotors
taata box -30-40
more sequences where pole binds and has enhancers upstream and downstream
b- 1 RNA polymerase
transcription in eukaryotes
3 types of rna polymerase
transcription factors involved
enhancer sequence which activator proteins can bind. adaptor proteins- all activate and dna folds.
how is gene expression regulated in eukarya?
Polyadenytion
3’ poly (A) tail- to stabilise the mRNA
Splicing- removing on introns
capping structure added to mRNA:
addition of 7-methylguaosine (binds 5’-5’ phosphate at start)
added to mRNA to stop degradation (DONT DESTROY) polyo virus targets this
Splicing
spliceisome- multi protein complex, help bind RNA around the introns recognise consenus sequence.
cuts out the introns at either end and binds the 2 exons (ligase binds 2 nucleotides)
alternative splicing?
the same gene can produce slightly or different proteins depending on the introns or extrons used.
examples:
Drosphilla gene- grey always present, r/g/b only retain in certain transcripts (38) can be
Genome strucutres:
bacteria and archaea
-no histones (bacteria)
-dna is circular
-divided into genomic DNA, replicon
DNA (plasmids, megaplasmids)
-introns and exons (rare)
inteins (do a similar job to introns)
-HGT
-prophage
oriC in e.coli
(bacteria)
initation
(whole genome)
definition of replication begins at unwinding
Opening of the strands to allow replication to begin
-one region binds single stranded dna the other double stranded
DUE- dna unbinding site full of a’s and t’s seperates from each other creating a ‘bubble’ and 2 single strands briefly.
- (2 replication forks)
- DNAa (enzyme) intitator protein, binds to the box site (double stranded) and binds to DUE (creating helix turn helix motif) binds acroos those sites and a second part of the enzyme which is an ATPase domain binds acrose DUE this winds itself up and seperates the 2 DUE strands.
- attaches to single strands, zooms along seperating the 2 strands, expensive of ATP.
Have seperate so enzymes can get in and a copy to need to be
seperated
-space between DnaA box site and DUE is important
-DnaA box site it binds to needs to have correct spacing
oric is a gene but doesnt make anything just facilitates
oriC in the Archaea
-same kind of size
-3-4 oriC around the genome
-Orbs: origin of replication boxes all found in 1-4 oric locations
what happens?
-initiator protein (orc1) binds to one of the oriC. It will bind well to oric number 1 and particarly to all the other oric.
-in some archaea also have WHiP proteins which do the same sort of job and can also act as initiators.
-many molecules of orc1 binding to oriC one will join together forming an=oligimorise
this then does the job of DNAa and does the unwinding
example:
saccharlobus solfataricus (wu et al)
oriC2
orc complexes bind to 1 and partically to another.
theory to multiple if you do it in 4 times then it speeds up time.
protein synthesis
123 are the current polypeptide chain that is enlongating→bound to tRNA in p site→new charged tRNA comes into the A site and has a complimentary anti codon→bond between 3-4 begins and ribosome shifts so that the og goes toward the exit site but attached to the chain as another aa and then cycle repeating (sliding of small thne large sub unit)
accesory molecules that aid this:
tRNA is linked to elongation factors not just floating around by itself (different in bacteria and eukarya)
GTP attached to trna and elongation factor
linked to an 1st initator factor bound to GTP (energy) to break down bond and new formation→Hydrolysis of GTP to GDP (release of a phosphate→ breaking high energy bond) this energy changes confirmation of ribosome to form the new bond
2nd initaitor factor- hydrolysis of GTP to GDP creates energy that for change of confirmaton (sliding of 2 subunit) formation of new bond and exit of the last one.
initiation of translation in eukayroitc cells
Monocistronic mRNAs: info for only one type of protein unless alt splicing
transcription (nucleus) and translation(cytoplasm) are seperate in space and time
Transcription can occur not in cytoplasm- for mitocondria
small ribosomal sub unit interacts with the elongation factors, charged tRNA, this is helped by the 5’ cap this complex is at beginning of mRNA (Small subunit only)→All starts scanning to find the initating AUG→ release of elongation factor→hydrolysis of ATP→ADP this energy recruits the large sub unit = complete machinery → chain elongation begins
where can gene expression be controlled?
*transcription (will it be transcribed or not how much if so)
*mRNA- processing (splicing etc)
*transport in messenger RNA
*where will mrna be will it be active
*translational regulation (makeup protein or not)
*Will the protein be used, how long will it last, hydrolysis right away?
*phosphorlaytion, ubiqylation etc
packing
