MODULE 1 BIO1007 notes

The central Dogma of life

 
Outline the central dogma of molecular biology and describe the information flow between DNA,
RNA and Proteins 2.
 
DNA-> RNA is transcription --> protein is called transaltion
 
DNA-->DNA is called replication
 
RNA--> DNA is called reverse transcription
^viruses with mrna genome rather than dna ( need to reverse transcript rna to DNA through enzyme
called reverse transciptase.
 
CANNOT go from Protein to RNA or DNA To protein or protein to DNA
 
 
 
 
Define the genome, transcriptome and proteome and how they differ from cell to cell.
 
The genome (DNA)
 
 Double stranded helical 5 prime to 3 prime and other strand opp
 Stable call can repair cytosine deamination
 Same dna in all cell but some may kick out some of it like red blood cells.
 Each cell uses only specific part of genome through transcriptome and proteome
 Differ to different stimuli
 Most Prokaryotes have circular chromosomes (bacteria archea) some have plasmids
 Very small (12kb-15MB)
 Mycoplasma is the one with the smallest amount of base pairs.
Eukaryotic
 (10Mb-150GB)
 Linear chromosomes
 Condensed into chromatine
 Wrapped around histone proteins.
 Humans have 6 billion bas pair , 22 pairs of chromosomes +sex chromosomes
 One from mother one from Father.
E.g Marbled lungfish 130 billion base pairs
 
 
Transcriptome (RNA)
 
Messenger RNA ( encodes protein sequences ) , makes multipple copies of mrna for each gene and
then it is designed to get degraded
 This is why it can tolerate cytosine deanimation to uracil ( the thing doesn’t get passed on it
degrades)
 Degradation occurs through ribose
  Its is ok if it is less stable than DNA
 Production rate varies you may only need some copies of a certain gene
There sre also ribosomal rna
Transfer rna
Small nuclear rna
 
Proteome ( Protein)
Mrna-protein-folding- used for below
 
 Ion channels
 Receptors
 Antibodies
 Enzymes
 Transcription factors ( bind to DNA help in making messenger RNA)
They make up 50 % of cell in dry weight
 
Proteins give the cell its shape, they form receptors, enzymes, hormones and growth factors, toxins,
transporters and antibodies.
 
 
 
The expression of some genes is altered by chemical modifications of DNA and proteins but NOT zto
the DNA itself -epigenetics
 Can be passed through generations
 
 
3. Appreciate the difference in size and construction between bacterial and eukaryotic genomes

The Central Dogma of life part 2

 
Define the universal genetic code (triplet/nonoverlapping/common)
 
Rna has uracil for thymine
 
20 amino acides need to come out of 4 bases
 
Single code of AUGC is not enough to code 20 diff amino acids only 4 possibilities
Double makes 16 possible pairs (still not enough)
Triplet -64 possibilities but there will be double of some amino acids but that’s life
We have a non overlapping system
These are called codon ( three bases that represent an amino acid)
Always read from 5 prime to 3 prime from mrna
STOP CODONS
UAA
UAG
UGA
START CODON
AUG
 
The genetic code is dgenerate or redundant ( had multiple for one)
Used by all life forms ( some minor exceptoons
 
 
 
2. Understand the concept of reading frames for translating a nucleic acid sequence into a protein
sequence
 
If you start reading the code not in three at first like two first then the entire code gets messed up
and doesn’t make sense.
 
The correct reading frame is the one that has a start codon, no start codon no priotein

 
 
 
The start codon determines the reading frame .
Missing base/deletion mutation causes entire code to be read wrong . Also know as frame shift
mutation.
Sometime will work out becomes same code for some amino acides ( Silent mutation)
Single point mutation/ single amino acid is changed ( one base is changed ) - may affect protein
function
 
 
 
3. Understand how to read
a genetic code table and translate nucleic acid sequence into protein sequence using such a table.
 Copying DNA
 
 
1. Outline the general mechanism for copying DNA to DNA before cell division -replication.
 
Always copying from 5 prime to 3 prime. ( so connects to 3 prime side of DNA)
Use nucleotide triphosphates as substrate
Add the nucleotide monophosphate to the 3’OH end of the growing chain
Form a high energy phosphodiester bond – Release pyrophosphate (PPi) – Uses a DNA
polymerase Need a primer (short piece of DNA/RNA) to start
 
-Pyrophospahre is released and breaks down from three to two phospates ( gives energy)
 
DNA polymerase
Make a DNA copy from a DNA template
• Need a primer to start
• Use deoxynucleotide triphosphates (dNTPs: dATP, dGTP, dTTP, dCTP) as substrate
• “Proof read” the last nucleotide added; often have 3’ to 5’ exonuclease activity to remove a
mismatched nucleotide
 
 
Both strands need to be copied
 

1. Start with parental strand

2. Unwind DNA to copy it
3. Made two complementary copies
 
 
2. List the unique problems associated with replication and describe the strategies used by
the cell to overcome these, including unwinding DNA, and leading versus lagging strand
replication.
 
 Long helical structure is hard to unwind, you get supercoiling
Topoisomerase enzymes cut strands allows to unwinds and stick back together (religate)
 
In cells only unwind small sections of DNA at a time
 
Copy DNA when cells are about to divide

Origin Sites (ORI) and Initiation

 Occur at AT ( bases) rich areas as its easier to pull strand apart because less stable
 Multiple sites bound by DNA binding proteins
 DNA helicase unwinds DNA
 DNA topoisonerase/gyrase stops supercoliing
 Replication forks form at either end
 Single stranded binding proteins coat singe stranded dna to keep strands apart and portect
DNA as it is now vulnerable ( because it is exposed)
 
Different methods for the leading and laggofn strand
 Primase put rna primer , dna polymerase sees primer and then starts replicating
 when doing lagging strand the DNA polymerase can only go the opposite way ( see
diagram )
 So what happens is more primase comes and more dna polymerase III and same thing
happens but in fragments called Okazaki fragments, the rna in between these fragments Is
then eaten up by a different DNA polymerase called DNA polymerase I. so now theres long
strands of D
 
 NA but not connect. DNA ligase connects them.
 
 

 
 

 
 
 
3. Describe the general functions of proteins that are required for DNA-replication
 
 
 
Eukaryotic replication
The structure of a nucleosome consists of a segment of DNA wound around eight histone
proteins and resembles thread wrapped around a spool. 
Multiple origins of replication on each origin
 
Special mechanisms (telomerase) for the ends (telomeres) of the chromosomes
Copying DNA part 2
 
 
1. Outline the general mechanism for copying DNA to RNA - transcription.
 
RNA polymerase
-Don’t need primer ( primer is a type of rna polymerase)
-Use ribonucleotide triphosphates (NTPs:ATP, GTP, CTP, UTP) as substrate
 No proof reading, rna lots of copies are made and gotten rid of unlike DNA
 
 
 
 
 
2. List the unique problems associated with transcription (including unravelling DNA, and making
multiple copies of small sections of the genome at different frequencies) and describe the
strategies used by the cell to overcome these.
 
Dna is the neitre genome
Only a small section of the genome is transcribed
These sections need to be transcribed thousands of times
Some sections need to be copied many times in one cell and not much in another
Where do you start and where do you stop ?
How do you switch it on off or upregulate?
 
 
RNA polymerase binds to a region of DNA called PROMOTER further up the 5 prime end
Its stops at the terminator region
 
Only one strand is transcibed each time can be either side
 
Transcription Initiation I
 
1. Transcription factors (sigma factor for bacteria ) are proteins that recognise a specific
sequence. This region is at -35 to -10 after that is +1 transcription site.
2. RNA polymerase is actually a subunit of enzymes that asscoiated with the dna bound
transcriptin factor
 
 
3. Describe the general functions of proteins that are required for RNA transcription 4. Compare
and contrast the differences between making DNA and RNA
 
In bacteria this sequence is known as the consensus sequence. There are 2 regions; at - 10 and -35
 
The -10 section is easy to melt ( pull apart) has a TATA sequence
 
Transcription Initiation II
 
Transcription factor leaves, the dna is pulled apart and rna polymerase starts transcripting.
 
Transcription Elongation
 
The 2 strands of DNA reanneal once the RNA polymerase has passed by
 
“transcription bubble” as the RNA polymerase complex moves along. One of the components in the
complex unwinds the DNA
 
This process is very fast in bacteria - ~50 nt per second
 
Transcription Termination
Transcription uses a signal encoded in the transcribed RNA to stop – a G/C rich sequence, often
followed by an A/T rich sequence
 
G/C rich region can form a ds hairpin structure which pulls the strand off the rna polymerase

OR
 
a Rho protein binds, and uses helicase activity to travel up to and dissociate the DNA/RNA hybrid
complex (physically pull or force apart
 
 
Gene expression regulation
 
Genes expressed at diff frequencies
 
Promoter strength
 DNA sequence optimised for storng or weak sigma factor rna poly binding
 Strong binding = more RNA copies made
 Weak binding = fewer RNA copies made
 
Repressors or accelerator ( specifically bacterial transcription )
 
 
Repressors
 
A protein repressor binds. This blocks the binding of the sigma factor/RNApol complex. They bind to
the promotor region
 
Accelerator
 
A Transcriptional Activator, a protein, binds at a specific DNA sequence and alters the structure of
the promoter so the transcription factor can now bind more frequently
 
This happens when a Weak promoter, DNA sequence is very unlike the consensus sequence
 
Both repressors and activators can be modulated by small molecule binding (often metabolites -
bacterial species)
E.g
Trigger change to activate activator
Binding relieves repression allowing sigma factor/RNA pol to bind

Protein Synthesis Translation

 
1. Explain the unique problems associated with converting the information as a nucleic acid
sequence to an amino acid sequence.
 
The need to convert a sequence of nucleotides to a sequence of amino acids
• The need to have the correct order of amino acids.
• Peptide bond formation is very thermodynamically unfavourable
 
There are three types of mrna that are used
Messenger ,
Transfer rna :matches the correct amino acids to the template
Ribosomal rna : combines with proteins to form the machinery for protein synthesis/catalyses
peptide bond formation
 
 
There is a ribosome binding site A bit of extra sequence in the mRNA helps to identify the start
codon.
 
T rna has an anitcodon which connect to start codon
Different tRNAs for each amino acid/codon combination
 
How does the t rna know which amino acid to get …
Different aminoacyl-tRNA synthetases for each tRNAs amino acid combination
Aa-tRNA synthetases
• Catalyse the activation of amino acids
• Use ATP hydrolysis to get the energy to make a high energy bond
• Attach the correct amino acid to its matched tRNA
• Recognise the anticodon and other parts of the tRNA
 
 
2. Outline the unique problems associated with protein synthesis, with particular reference to the
unfavourable thermodynamics of peptide bond formation and the requirement for order. Describe
the strategies used by cells to overcome these problems.
 
This reaction is extremely thermodynamically unfavourable due to the large amount of water
around. Hydrolysis is always favoured over condensation in an aqueous environment (Equilibrium
L7A)
Protein Structure
 
Describe the differences between primary, secondary, tertiary and quarternary structure of
proteins.
 
Primary Amino acid sequence
• Secondary Local structures § Alpha helix & Beta sheet
-backbone hydrogen bonding interactions are very important
Sidechain interactions help hold the structure together and form the tertiary structure
• Tertiary Overall 3D arrangement of a polypeptide chain
 
• Quaternary Organisation of subunits (Many but not all proteins have multiple subunits)
 
 
2. Appreciate that the protein sequence defines the protein fold and function, and that protein
molecules are held together by the combination of many bonds.
 
 
3. Demonstrate how 3D protein structure is related to function using the example of the alpha
helix in DNA binding proteins.