Cell theory & molecular mechanism of DNA processes

What key features comprise a cell

Genetic systems allowing hereditary, replication & metabolism, compartmentatlisation

What is cell theory

All organisms are composed of one or more cells

Cells are the basic unit of life

All cells arrive from pre-existing cells —> leading back to LUCA

Theory of how life evolved in the molecular soup

Hostile volcanic environment, frequent storms including UV light and lightning that would have been catalysing reactions of atmospheric gases

Key biological precursor molecules like amino acids, sugars, nucleobases form and fall into oceans

Condensation by chance into polymers

Possibility of a polymer catalysing own replication

Self-replicating polymer would dominance molecular soup and begin evolving

Problem with DNA as the first self-replicating molecule

DNA cannot replicate itself as it requires protein catalysts

Could RNA be the first self-replicating molecule

Is not strictly self-replicating as it requires another RNA molecule to act as a template, but one RNA can make a copy of another

Catalytic RNA (ribozymes) can catalyse synthesis of a new RNA molecule

2 problems with making RNA in a molecular soup

Dilution very weak, chance of two nucleotides meeting to form a chain eventually forming RNA is very small

RNA is unstable in water. Ocean favours hydrolysis and condensation is needed to convert nucleotides to RNA

Theory of where RNA could have originated

Clay mineral layers

Why would clay mineral layers be the perfect site for prebiotic RNA

All the molecules needed to make RNA (nucleotides, nucleobases, phosphates, sugars) bind to surface of clay-silicate minerals and become concentrated together in the layers - greater chance of molecules meeting

Repeated cycles of wetting and drying, condensation becomes favoured

Problems with RNA world theory

Dependence on the wet-dry cycle, really really slow

Lots of molecules, especially lots of sugars, would be present in primordial soup —> why is RNA made out of ribose specifically

Only ticks the box of hereditary, unclear how RNA would lead to cell membrane, metabolism, and cells 

What are membranes made out of and how

Amphiphile molecules - molecule with both polar and non-polar properties

They are self-assembling, spontaneously form in the presence of water (which is polar) 

Types of membrane structures

Micelles, vesicles, bilayers

Could phospholipids have formed prebiotically

No too complex, but simpler amphiphiles may have been present and self-assembled into vesicles and micelles

Evidence of membranes forming

Experiment where nucleotides and lipids heated and concentrated until total dehydration micelles formed and then fused with one another to create multilamellar films with nucleotides inside

Then after dehydration and rehydration cycles RNA molecules were formed, ending up with RNA inside vesicles

What conditions are needed for spontaneous metabolism

High temperature

High pressure

Redox active metals to act as catalysts (e.g. iron or nickel)

Why are alkaline smokers thought to be potential sources of life

They form due to exposure of the mantle to seawater, reaction called serpentinisation occurs forming new minerals - with minerals like iron, nickel

The reactions generates heat, hydrogen gas, hydroxide ions

There is high pressure at the bottom of the ocean

Sets up all the conditions for spontaneous metabolism - between hydrogen gas and carbon dioxide dissolved in the water

How are hydrogen gas and carbon dioxide made to react

CO2 reduced to organic hydrocarbons by the proton gradient, capturing electrons from H2. FeNiS and FeS act as catalysts

Proton gradient needs to be established so enzymes can capture electrons, done using an inorganic Fe/Ni wall with acidic seawater on the outside and alkaline hydrothermal fluid coming up

Problems with the alkaline smokers origin theory

Self-assembling molecules eventually form, wouldn’t they stop molecules interacting with the metals?

How did this basic chemistry evolve into non-enzymatic metabolism and complex molecules?

Possible process of cells forming from primitive membranes with amino acids and FeS crystals

Amino acids bind to FeS crystals

Some amino acids would be hydrophobic, could embed in cell membrane

Then act as catalyst to produce the proton gradient across the cell membrane, driving the reduction of CO2 into hydrocarbons and more amino acids and fatty acids produced

Fatty acids produced would increase the cell membrane, at one point would divide into two due to the size —> cell replication

Cells with the best catalysts (fastest growth) ‘selected’ for

Central dogma of molecular biology

DNA transcribes to RNA which translates to protein, DNA self-replicates

RNA world theory

Hypothetical stage in evolutionary history, there were self-replicating RNA molecules before DNA and proteins evolve

Why is RNA called an acid

The phosphate can donate hydrogen ions and become negatively charged

Bases that RNA can have (and their nucleoside forms)

Adenine & adenosine, guanine & guanosine, cytosine & cytidine, uracil & uridine, and thymine & thymidine

Why could nucleotides have prebiotic origins

Phosphate is a common mineral, ribose could potentially form from formaldehyde in a formose reaction, and purines can be formed from polymerisation of cyanide

Key thing to tell which base is which when looking at chemical structure

1 or 2 carbon rings denotes purine (G and A) vs pyrimidine (U and C), and the number of hydrogens each molecule can donate/accept tells you the number of hydrogen bonds that can be formed

Which direction is RNA read and synthesised

From 5’ to 3’

Unique property of RNA (that DNA doesn’t have)

It has a 2’OH group that can both accept and donate hydrogens for hydrogen bonds, allows RNA to fold into lots of different combinations.

And the 2’OH group can bind to proteins in the various RNA functions

Does bonding have to just be between A and U, and C and G in RNA

No, for example in tRNA there are G and U base pairs

What does the variable loop do in RNA

Allows for flexibility and conformational change

What defines whether something can form life

Store information

adapt/mutate,

sense the environment,

catalyse reactions,

replicate itself

RNA sensing the envionment

It can sense ions, temperature, pH, salt concentrations etc.

Metabolites bind to riboswitches in RNA sequence and regulate gene expressio

RNA catalysing reactions

Ribozymes are enzymes based on RNA

Also have RNA-based ligases and polymerases

Can RNA replicate itself

Probably yes but no solid evidence yet. RNA-based ligase found that can link its own precursor RNAs together to create itself

What is the evidence for an RNA world

relics

What are the ‘relics’ of the RNA world

Cofactors essential for reactions and metabolism like FAD, CoA, NAD, ATP have adenosine as a base — suggesting it was a conserved element. RNA used to make proteins, is part of ribosome

Formation of proteins - levels of their structure

primary - chain of amino acids/residues

seconary - folding into a-helixes and b-sheets

tertiary - more folding due to proteins seeking lowest energy state

quaternary - multiple protein subunits arranged together

Bonding between amino acids

Peptide bond between carboxyl and amino group, formed in condensation reaction.

leads to partial double bond at both C=O and C=N giving it resonance structure, and peptide bond cannot rotate.

What is the secondary structure determined by

Steric hindrance (interactions of atoms in the molecule)

Four main interactions in the tertiary structure, and how do they fold

Hydrophobic effect, disulphide bridges, hydrogen bonds (between polar amino acids), ionic bonds (between charged amino acids)

Fold themselves or use chaperones

What is conformational change

Change of the tertiary structure and active site due to initiation, e.g. substrate binding in induced fit model

What is allosteric regulation

Binding of molecule at an allosteric site to assist uptake of substrate

What are often used as cofactors

metals

What can occur to proteins after translation

PTMs - over 200 types, reversible or not.

e.g. phosphorylation, nitrosylation, acetylation

Why are proteins better catalysts than RNA

Smaller molecular machines w/ more chemistry and detail - 20 amino acids compared to 4 bases of RNA, so better for catalysis

Downfall of proteins (compared to RNA and DNA) and why

Cannot replicate - no method to recognise different residues

Proteins can only make peptides - using non-ribosomal peptide synthases

Structure of mRNA into reading frames etc.

RNA has open reading frames (translates into functional protein) and untranslatable regions.

Three reading frames in mRNA - correct one identified using start and stop codons

Initiation sites for transcription in euks vs proks

Eukaryotes - frame begins with first AUG from the 5’ end in Kozak sequence

Prokaryotes - frame begins with AUG after Shine-Dalgarno sequence

Special property/bonding of the third base in the codon - why

Can change but still code for the same amino acid

Exhibits ‘wobble pairing’ with the corresponding base on the tRNA anticodon which can act as a ‘wobble' base’ - can move to pair with multiple bases and mRNA codons

Important as it reduces number of tRNA molecules needed

Difference between euks and proks - how many proteins genes code for

Eukaryotes - mRNA is monocistronic, each gene codes for one protein. Allos for greater regulation and diversity.

Prokaryotes - mRNA is polycistronic, genes can code for multiple proteins under same promotor and operator

Different types of mutations

Silent - doesn’t change aa sequence, change third base in codon

Missense - changes aa sequence, either first or second codon

Nonsense - cause premature termination by creating stop codon

Frameshift mutation - either insertion or deletion

Steps for tRNA biogenesis

tRNA precursors are often tandem arrays of different tRNAs, transcribed and then cleaved by RNAse P at 5’ end, and RNAse D at 3’ end

tRNA is then modified, including addition of CCA at the 3’ end - by tRNA nucleotidyl transferase, and base modifications by small nucleolar RNAs

Sometimes spliced

Structure of tRNA

Clover shaped secondary structure. Anticodon on the middle loop (one base being the wobble base).

The CCA at the 3’ end carries the activated aa

Variable loop that can change shape/twist to give some variety

Charging of tRNA / becoming carrier

Done by aminoacyl-tRNA synthase (aaRS)

1. Amino acid is adenylated by addition of ATP (pyrophosphate leaves, with H2O, making it condensation)

2. Amino acid transferred to the 3’OH of tRNA, releasing AMP

aaRS can detect if wrong aa is added, will hydrolyse bonds and start over

Structure of ribosome

Large subunit - containing peptidyl transferase, that forms peptide bonds

Small subunit - containing mRNA guide

Mixture of protein and RNA

Eukaryotes size is 80S, prokaryotes size is 70S

How many tRNAs and mRNAs are in the ribosome at one time

3 tRNAs at different sites.

A site - tRNA carrying in amino acid

P site - tRNA linked to growing aa chain, peptidyl-tRNA as peptide bond formed

E site - empty tRNA exits ribosome

One mRNA bound to ribosome

Three steps of translation in prokaryotes:

Initiation

1. shine-Dalgarno sequence recognised binds to rRNA on small subunit.

2. fMET-tRNA binds to AUG start codon, bringing in Met

3. Large subunit binds, placing fMET-tRNA in P site

Elongation

1. acyl-tRNA (tRNA with amino acid) loaded into A site

2. Residues from tRNAs in A and P site close causing peptidyl transfer and peptide bond formed

3. Ribosome shifts one codon in 3’ direction - peptide tRNA enters P site, and empty tRNA exist from E site

Termination

1. Release factor binds to stop codon

2. peptidyl-tRNA hydrolysed and product released

3. Both subunits and tRNAs dissociate from mRNA

RNA world relics in the production of proteins

Core activity done by RNA - mRNA, tRNA, snoRNA, rRNA, RNAse P etc.

Problem with RNA - why DNA needed

RNA generally stable but mutates spontaneously - C to U and cannot be repaired by RNA

Need for stable storage, that can encode all information

Why is DNA more stable than RNA

Has deoxyribose instead of ribose

Has no 2’OH so A=T and C=G always bond, no exceptions unlike RNA

DNA has an extra methyl - base T instead of U. So if C deaminated to U it could be easily detected

How are the building blocks of bases formed

Begin as nucleoside diphosphates

Ribonucleotide reductase removes 2’OH from NDP and kinase adds the 3rd phosphate to form deoxynucleotide triphosphate (dNTP)

Ribonucleotide reductase makes forms for all bases - dADP, dGDP etc

RNA world relic in the formation of bases

dTDP made from dUDP - uracil (and RNA) existed first

DNA replication - importance of DNA polymerase

B-clamp keeps DNA polymerase on strand until whole replication occurs. Polymerase elongates the RNA primer, synthesises 5’ to 3’

What happens with a mismatch / mistake in DNA replication

If mismatch detected, DNA polymerase can reverse - reduces replication errors

If mismatch not detected and is synthesised, the mismatched section is removed and repaired with new nucleotides. Catalysed by various DNA polymerases, lastly ligase.

Lagging strand replication

Primase binds and produces an 11nt RNA primer using the DNA template

A different DNA polymerase synthesises Okazaki fragment (around 1000 bps), RNAse H degrades the RNA primers.

Another DNA polymerase extends the Okazaki fragments, and ligase joins them

Difference in DNA replication between eukaryotes and prokaryotes

Prokaryotes - replication is continuous, single origin of replication

Eukaryotes - only replicate in S cell cycle phase, can have multiple origins

How to deal with end replication problem

Telomerase binds to and extends the parental strand - using RNA template.

Makes a telomere extension using DNA synthesis

Primase produces an RNA primer, DNA polymerase extends the primer and ligase connects the phosphate backbone

RNA polymerase activity in transcription

Synthesises from 5’ to 3’ (like DNA polymerase) but uses dsDNA template

Starts from an initiation site, without a primer

Energy for mRNA synthesis and DNA unwinding comes from pyrophosphate release - condensation reaction forming the mRNA strand

Transcription initiation in prokaryotes

sigma factor of RNAP scans DNA and recognises two upstream motifs - Pribnow at -35 and TATA at -10

RNAP unwinds 17bp, creating a transcription bubble where RNA synthesis starts

Sigma factor dissociates, RNAP produces mRNA

Transcription initiation in eukaryotes

DNA has multiple upstream promoters - but core promoter is the TATA box (others are regulator elements, enhancer sequences).

TATA binding protein (subunit of TFIID) binds to TATA box, recruits TFIIA and TFIIB (transcription factors)

TFIIB recruits RNA polymerase II and TFIIF. TFIIE joins and recruits TFIIH (carries out helicase activity).

When upstream regulatory elements give the signal - transcription bubble forms and RNA polymerase II is phosphorylated

RNA polymerase II dissociates from TFIID and begins transcribing

What is on the 5’ end of transcribed mRNA after transcription - euks & proks

Prokaryotes - triphosphate purine (G or A)

Eukaryotes - 5’ cap - N&-methyl-guanidine-5’-triphosphate (required for splicing, translation, and stability)

Termination of transcription in prokaryotes

Can be rho independent or dependent

Rho-independent - termination signals in the 3’UTR of mRNA cause NusA to produce a hairpin (loop) that interferes with and terminates RNAP

Rho-dependent - rho binds to the rho-utilisation site (rut) and the collision of rho and RNAP terminates transcription

Termination of transcription in eukaryotes

polyA signal leads to cleavage by endonuclease downstream, terminating polymerisation.

Poly-A polymerase then adds a polyA tail to increase RNA stability

What process can happen after transcription - describe it

Splicing to remove intron sequences in the pre-mRNA

Bases in the intron react with each ohter, forming a lasso that removes itself from the RNA - leaving only exons.

Catalysed by the spliceosome (ribozyme) - formed of 5 small nuclear RNAs with proteins

List the extra regulatory steps for eukaryotes - done to mRNA

5’ cap, poly-A addition, splicing, export