Bacterial growth and cell division L4 Flashcards
what are the events required for cell division
- DNA replication
- Transcription of genes and production of new proteins
- Septation and synthesis of a new cell wall
- Cell separation
Events occur in a set order, can be divided into distinct periods of cell cycle
what is the integrated cellular machine
Energy can be generated in many ways – biochemical generation
Need to generate energy – all processes need energy to drive them
what is the bacterial chromosome structure of DNA
DNA supercoiled, not uniformly associated with proteins – compared with Eukaryotic chromatin structure
what is supercoiling
over winding of DNA - DNA has a natural structure it forms with a natural structure and amount of twisting – B form DNA
what causes the cell to be wound tighter and what is natural DNA like
Enzymes in cell either wind it tighter or under wind it - natural state DNA is tighter wound
what happens to DNA when start winding up
DNA up cannot unwind – physical thing that happens
where does DNA get energy and what is it used for
DNA has energy – wants to unwind back to relaxed state, energy helps transcription
Supercoiling energising DNA molecules
what other proteins are bound to DNA
- RNA polymerase
- Transcriptional regulators
- small DNA binding proteins
how many chromosomes do bacteria have
not all have one
what chromosomes do bacteria have
not all have circular some have linear
do bacteria require plasmids
cell can lose plasmid and still live
if bacteria lose this they die
If loose one of the circular molecules will die – as loose genetic information that is vital
Chromosomes – they have genes that cannot be lost from the cell
where does DNA polymerase ‘work’
DNA polymerase can only add bases to 3’ end of a primer
- once DNA is extended from primer DNA Pol I removes RNA
what does cell need to recognise needs to be replicated
origin of replication
where does DNA replication start
OriC on chromosome DnaA (initiator protein) binds to OriC
what does primase synthesis do
make RNA primer
what carries out DNA replication
DNA polymerase III (used for bacteria)
which way does DNA replication occur
5’ to 3’ direction on both strands
Leading strand DNA synthesis is continuous, carried out by DNA polymerase III
Lagging strand DNA synthesis is discontinuous
- Okazaki fragments are made by DNA Pol III
what is the DNA polymerase I function
- Extends end of Okazaki fragment until adjacent RNA primer reached
- 5’-3’ exonuclease activity of DNA pol I removes RNA primer
- DNA pol I then fills gaps between Okazaki and uses 3’-5’ exonuclease activity (proofreading function) to correct errors
how does a DNA replication fork in bacteria form
Helicase unwinds dsDNA ahead of replication fork
SSB = keeps unwound strands apart
what is the DNA elongation rate
Proceeds at a constant rate irrespective of growth conditions
where does DNA elongation occur in the cell
at cell centre by a complex of proteins = Replisome
when does termination occur in DNA replication
Two replication forks meet at terminus (terC)
A termination protein (Tus) is bound to terC region
terC is flanked by DNA sequences, form structure that block replication forks progression
Ter sequence has sequences either side of it, slow the DNA synthesis and replication down
what is partitioning
separate chromosomes into two daughter cells
how can DNA replication be imaged during partitioning
Fluorescent in situ hybridisation (FISH)
Uses Fluorescent probes specific for DNA sequences of oriC & terC
Cells viewed using fluorescent microscopy
what does transcription require
RNA polymerase holoenzyme consists of six protein subunits
- Core enzymes (five subunits)
- Sigma factor
where do sigma factors bind to for transcription
binds to the -10 and -35 sequences in bacterial promoters and identifies beginning of gene
how do regulatory proteins work to create RNA
Regulatory proteins control RNA pol binding to promoters
Any negative numbers not incorporated into RNA
why dont bacterial mRNA undergo significant processing
- Primary transcript synthesized by RNA pol is mature mRNA
Transcription and translation both occur in same cell compartment
Translation usually begins before transcription is complete
what are the three phases of bacterial translation
- Initiation – binding of ribosomes to mRNA
- Elongation – movement of ribosomes along mRNA, synthesis of polypeptide
- Termination – release of mature polypeptide, recycling of ribosome
what occurs during prokaryote initiation
ribosome binding site
Ribosomes usually bind to mRNA as separate subunits
- 30S binds first
- IF3 (initiation factor 3) binds to 30S unit & prevents binding to 50S
fmet-tRNAmet binds to 30S at P (peptidyl) site
- requires IF-2 (initiation factor 2) and GTP
- f-met tRNAmet is the only tRNA that can enter the P site
30S + fmet-tRNA binds stably to mRNA
- requires IF-1 (initiation factor 1)
what does translation need to start
translation need methanonine that’s modified with formaldehyde
GTP energises the process
Only f-met tRNA can fit into P site – only tRNA that can start
how and when does prokaryote translation termination occur
Termination occurs when STOP codon is in A site
Requires specific Release Factors to cause dissociation of ribosome complex
what are examples of different stop codons recognised by protein release factors
- UAA (Ochre) RF1/RF2
- UAG (Amber) RF1
- UGA (opal) RF2
- stop codon P site must be occupied
- RFs act at A site
what are polyribosomes
Multiple ribosomes bind to one transcript
polyribosome function
multiple copies of new protein being produced from one RNA molecules
what happens in cytokinesis
Nucleoid and cytoplasmic contents divided between two daughter cells
Cell Envelope (cell wall plus cytoplasmic membrane) invaginate
Directional shift in growth of peptidoglycan occurs at a predetermined position by the in-growth of a septum
Cell membrane and cell wall – take part in slightly different things
what is an essential protein for cells
FtsZ
what is FtsZ and septum formation
Boa constrictor FtsZ is polymerised into chain GTP is hydolysed by unknown trigger Protein chains begin to constrict cell Cell wall shows elongated cells with rings present
how is FtsZ imaged
using FISH for different cell components
FtsZ labelled green, located at replisome, know this as the DNA – nucleiod, is the dividing cell with a chain of FtsZ between it – one cell that’s been pinched and splitting into two daughters
how is cell wall imaged
using fluroscent microscopy
how does the nucleoid form from cell wall and FtsZ
FtsZ pinching off, finish cell separation need peptidoglycan making two cell walls
how do cells grow and divide
binary fission
how is the time course of helical and ring structures in peptidoglycan revealed
staining newly synthesised PG with fluorescently labelled Vancomycin (antibiotic than binds to new PG)
Bands of new peptidoglycan as it is growing, get helical rings
what is formed depending on whether separation is complete or not
Separation may be complete (single cells released) or incomplete (chains or clumps produced)
what forms if cell divides in same plane twice
chain
what forms if cell divides in two different planes
tetra
what causes the cell division in same or different planes
is genetically predetermined
what replication occurs on plasmid
OriV
what is a limiting factor in determining shortest generation time
DNA elongation
what happens in prokaryote protein synthesis
- elongation
- aa-tRNA entry mediated by EF-Tu (elongation factor Tu)
- forms 3° complex with GTP
- delivers tRNA then leaves ribosome
- hydrolysis of GTP to GDP occurs during release
- EF-Tu is recycled by EF-Ts - peptidyl transferase activity is located in 50S subunit
- peptide bond formed, ribosome changes conformation allow movement of tRNAs
- translocation moves tRNA to P site; uncharged tRNA leaves via E (exit) site
how is prokaryote elongation acheived
further charged tRNA molecules entering the A site (aminoacyl site) & formation of peptide bond
what does peptidyl transferase activity need
rRNA as part of catalytic site
what does conformational change of ribosome in prokaryote protein synthesis require
requires GTP & EF-G (elongation factor G)
