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Biomedical Science Microbiology module summary
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Biomeducal Science course. 2nd year Microbiology module suymmary notes for final exam
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DNA REPLICATION:Streptococcus pneumoniae - capsule secreting smooth colony morphology (virulent) or non-secreting,
rough colony (non-pathogenic) - newly transformed bacteria (R from S) have acquired information to
make capsule from the heat killed cells - information replicated during replication. - transmission
occurred during cell division. - variation existed between capsule producing strain (s strain) and non-
capsule (R strain). conclusion - genetic material from heat-killed S type had been transferred to living R
type and provided a new trait.
Avery, Macleod, McCarty 1944: purified DNA< RNA and protein from type S bacteria and added it to the
R strain. Initial experiments suggested that purified DNA extract converted R strain to S strain.
Problems - purification by nature will always contain some contaminants. could be argued that the
contaminant is the genetic material.
Hershey and Chase 1952 - DNA genetic material of T2 bacteriophage - bacteriophage contains
blueprint to replicate itself in a living host - Needed a way to distinguish T2 protein from T2 DNA -
radioisotopes: 35S used to label proteins, 32P used to label DNA - mixed labelled phage with E. coli
host.
Erwin Chargoff - isolated DNA, measured the levels of the 4 nitrogenous bases - found that the level of
A to T and C to G was the same - suggested that DNA was made up of equal quantities of purines and
pyrimidines.
Initiation and unwinding - assembly of a replication fork - fork is generated by a complex of proteins -
e.coli mechanism established by in vitro studies - major initiator protein is the dnaA protein - recruits
DNA helicase (dnaB) and DNA primase (dnaG) proteins - helicase bound by an inhibitor protein (dnaC),
which is released to allow DNA binding at replication origin - helicase begins to unwind DNA
DNA helicases – break the H bonds that join the complementary bases – SSB protein – stop the
unwound DNA from re-joining – Topoisomerases (DNA Gyrase) – reduce the torsional strain caused by
the unwinding of the double helix
Primer synthesis - marks the beginning of synthesis of the new DNA molecule - primers- short stretches
of nucleotides synthesized by an RNA polymerase - primers are required because DNA polymerases can
only add deoxyribonucleotides to the 3’-OH group of an existing chain and cannot begin synthesis de
novo - after elongation is complete, primer removed and replaced with DNA nucleotides.
Pol I – 928 amino acids – adds the correctly paired nucleotides to a template strand – catalysed
synthesis of DNA in in vitro when provided with all 4 deoxynucleotide-5’-triphosphates, template strand
and a primer. Incoming dNTPs form H bonds with the template strand, DNA Pol breaks bond between 1 st
and 2nd phosphate in dNTP releasing pyrophosphate
Properties of DNA pol – bases are selected within DNA Pol active site by complementary base pairing
with template strand -- chain growth is in the 5’ to 3’ direction and is anti-parallel to template – DNA
Pol cannot initiate DNA synthesis de novo
DNA replication – DNA strand at origin of rep creates 2 forks – primers initiate synthesis, 1 st Okazaki
fragment is made in opposite direction for lagging strand, leading strand elongates and 2 nd Okazaki
fragment made , 3rd Okazaki fragment made and 1st + 2nd are connected together via DNA ligase
Termination at Ter sites requires action of Tus protein, bound Tus proteins prevent strand displacement
by helicase, the orientation of the protein is what blocks the replication fork.
, Eukaryotic DNA replication enzymes – MCM, RPA, RNase H1, Flap endonuclease-1 , PCNA
Prokaryotic
equivalent for
DNA
replication –
initiation
complex,
single-
stranded
binding
protein, DNA
polymerase,
Sliding clamp
High replication accuracy – cells maintain balanced levels of dNTPs reducing mis-incorporation,
polymerase exhitis inactive and active forms which help position an incoming nucleotide, 3’-5’
exonuclease activity of DNA Pol detects and eliminates error
Mismatch repair system – MutS binds to mismatched base pair, MutL binds to MutS, MutS2MutL2
complex loops out DNA, new DNA strand is only partially methylated MutH endonuclease binds and
excises mismatched bases, DNA Pol III/I re-synthesises fragment, DNA ligase seals strand
RNA POLYMERASE AND TRANSCRPTION
RNA polymerase binds to a specific DNA sequence in front of the gene - the promotor – it attracts RNA
polymerase to the gene and tells the enzyme that a gene is nearby
GENE EXPRESSION:
Polymerase II - they form a large complex known as the basal apparatus, located in the nucleoplasm -
Pol I - located in the nucleolus - Pol III - located in the nucleoplasm - A typical gene transcribed by RNA
polymerase II has a promoter that extends upstream from the site where transcription is initiated.
Transcription initiation and the basal apparatus - Pol II and associated TF will be recruited to the core
promoter near the transcription start site - TATA-binding protein initiates the preinitiation complex
assembly by binding to the TATA box at the promoter - TFIIA and TFIIB interact with TBP and reinforce
its binding to DNA - TFIIB recruits RNA Pol II and TFIIF over the transcription start site - TFIIH mediates
‘melting’ of the TSS to form the open complex that is stabilised by TFIIE - TFII proteins are ‘basal’
transcription factors sufficient for basal transcription only
Core Promoter - can overlap/include the transcriptional start site binding site for TBP/TFIID, Pol II, and
general/basal TF - Proximal/upstream promoter - lies 70-200 bp upstream, CCAAT and GC boxes, can be
of varied complexity depending on the gene, binds TF. act in coordination with the preinitiation
complex to aid RNA polymerase in initiating transcription and in regulating the frequency of
transcription.
CCAAT box - 70-80 bases upstream of the transcription initiation site, and about 150 bases upstream of
the TATA box - binds transcription factors -- GC box - GC-rich sequence motif, often found in proximal
promoter - binds SP!* (a TF first identified in SV40 gene transcription)
eukaryotic gene expression - each gene requires its own promoter and other regulatory elements -
controlled by large multi-subunit protein complexes which bind to regulatory DNA sequences -
activators required for activity above basal - tissue and cell-type specific regulation gene specific,
environment specific
Eukaryotic RNA polymerases - RNA polymerase is the enzyme that synthesises RNA using a DNA
template - each RNA Pol works in conjunction with its own set of basal transcription factors - required to
form the initiation complex.
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