Gene Regulation in Prokaryotes

Generally, larger the organism = larger genome size

Genomes of prokaryotes - smaller than eukaryotes

Prokaryotic genomes encode 1000-6000 proteins and are haploid

Compressed and packaged by DNA supercoiling

Localised in cytoplasm - transcription and translation simultaneous

Nucleoside - N-base + 5-carbon sugar

Nucleotide - nucleoside + phosphate

Nucleooside analogoues used as antiviral and anticancer agents. Activated in cell by conversion to nucleotides. Administered as nucleosides - charged nucleotides cannot cross cell membranes

2 grooves in DNA - major (22A) and minor (12A)

Template strand copied into RNA (T -> U) 

T only base pair with A, U base pair w/ others = better DNA fidelity (less mutations)

Coding sequences run in 5' -> 3'

Same DNA section contain additional coding regions on reverse strand running in 5' -> 3' direction

Reading framem - consectutive, non-overlapping triplet nucleotides equating to amino acids/stop signals in translation (CODONS)

Each DNA strand  = 3 possible codons

Operator + promotor regions - determine where transcription begins

Crick - deals with detailed residue-by-residue transfer of sequential information. Informarion cannot be transferred back from protein to protein/nucleic acid

SIMPLY:

• DNA makes RNA makes proteins 
• RNA transcript levels proportional to amount of protein synthesis - 1st form of gene expression control

mRNA - transcribes genetic code from DNA into protein making form - carries the message from nucleus to cytoplasm

rRNA - forms structual elements of ribosome that directs translation of mRNA

tRNA - transfers amino acids to ribosome

Promoter region - where RNA polymerase binds

Operator - site next to promoter where regulatory protein binds

Coding sequence - for protein or RNA and flanked by start (ATG) and stop codon

Terminator - RNA polymerase ends transcription

Bacterial core - 4 proteins (abb’w), forming 5 subunit complex (a2bb’w):

• a dimer - regulatory role in initiation
• w dimer - assembley 
  
• b + b' subunits - form active site
  

RNA polymerase holoenzyme - additional sigma factor to recognise promoter region in initiation

Slides along DNA to search for promoters

• Specific binding - binds to promoter regions
• Non-specific binding - binds to non-promoter DNA

E.coli - several sigma factors recognised by RNA polymerase occuring under different conditions. Each different cell has housekeeping sigma factors - esssential genes and pathways operate (70 in E.coli)

Holoenzyme - core polymerase and sigma factor (needed for initation of RNA synthesis)

• Initiation - binding of RNAP holoenzyme to DNA
• Elongation - open complex formation (transcription bubble), DNA unwinds = single strand w/ active site
• Termination - termination signal in DNA forms RNA haripin in emerging transcript = polymerase released

• Sigma factor associates w/ core RNA polymerase
• Searches for promoter site
• Recognises promoter consensus sequence
• Promotes local unwinding of DNA = expose template strand for copying (starts at Pribnow box - low H bonds)
• RNAP cannot form full length transcript until releases strong binding to promoter
• Repetitive synthesis/release of short RNAs - abortive initiation - accompanies steady-state transcription (~95% total RNA contains produces 2-16 bp)
• Sigma factor diassociates
• Promoter clearance - ensures propoer orientation of DNA/RNA polymerase complex to enter elgonation

• RNAP catalyses phosphodiester bond formation - link nucleotides to form RNA in 5'->3'
• 1 round polymerisation = 1 pyrophosphate (2 - P groups bonded)
• Substrates = ribonucleotide triphosphates

• When termination signal IN sequence of RNA transcript itself
• Palindromic hairpin forms followed by U bases
• Stem loop structure = RNAP pauses
• U bases after = unstable (less H bonds) 
• Complex dissociates

• Rho factor - ring like structure that binds around mRNA during transcription
• Attaches to utilisation (rut) site
• Moves along mRNA in same direction as RNAP
• Hairpin forms - RNAP pauses
• Rho catches up and contacts RNAP
• Rho pulls mRNA out of polymerase

Operon - functional unit with a group of genes transcribed from same promotor and controlled by same operator site/regulatory proteins

Regulon - set of genes (and/or operon) expresssed from seperate promoter sites, controlled by same regulatory molecule

Global regulons coordinate expression of many genes/operons and may induce some but repress others

Genes encoding for enzymes of metabloic pathways are grouped in operons on chromosome = coordinated regulation/gene expression of multiple genes at same time

RNA transcript codes for mutiple proteins

Regulatory sequence adjacent to unit determines transcription = operator

Regulatory proteins interact with operators to control gene transcription

Operators can be upstream, downstream or overlapping with promoter

Regulatory proteins bind to operator and influence access of RNAP to promotor - affect rate of transcription initiation

As mRNA made a ribosome will engage and begin translation process - prokaryotes have to nucleus

Rate ribosomes make protein depends on availablility of tRNA with appropirate amino acid

Example - trp operon

• Codes for genes involved in Trp synthesis
• Availablility of Trp low = operon active, producing Trp making enzymes (vice versa)
• Structure of RNA defining RNAs function - ribsosome involved

Trp conc high - trp operon off

• Concentration of tRNA^Trp  high
• Ribosome moves past site 1 - binds to site 2
• Prevents 2:3 base pairing
• Site 3 binds to 4 = 3:4 hairpin followed by Us
• RNAP falls off = no transcription

Trp conc low - trp operon on

• Concentration of tRNATrp low
• RIbsosome stalls at site 1
• RNAP makes 2:3 base pair to make stem loop = no 3:4 hairpin
  
• RNAP not stalled - carries on to complete transcript

Availability of tRNA controls transcription levels BUT codons themselves also used

Codon codes for single amino acid = 64 possibilities

Only 20 amino acids = more than 1 codon used for same amino acid

Not all tRNAs available at same level - organisms have preference for particular tRNAs

Some codons - low frequency use = likely ribosome will pause frequenctly = breakdown of ribosome/template complex

High frequency codons increase protein expression levels - effects speed of ribosome

• Faster rates along transcripts with higher codon adaptions to tRNA pools
• Effects in non-coding regions - alters RNA secondary structure

Codons can control protein folding as mRNA translation controlled spatially and in time

Transcription factor - proteins controlling rate of transcription (sequence-specific DNA binding factors)

Transcription factors bind to operator sites and enhance/inhibit RNAP binding at promoter site

2 parts - 1 part binds DNA 1 part binds signal (usually small mol)

trpR produces trp repressor - transcription factor

Unbound trpR not suitable conformation to bind to operator section

Concentration trp high - trp acts as corepressor and binds to trp repressor = conformational change

Allows repressor to bind to operator site

Prevents RNAP from initiating transcription - physically blocks it

Lactose can be another energy source when glucose unavailable. lac operon is 3 structural genes:

• LacZ - enzyme B-galactosidase cleaves disaccharide lactose into glucose/galactose
• LacY - membrane protein transporter = more lactose in cell
• Lac A - role unknown

lac operon regulated by transcription factor called CAP and internal metabolite is cAMP - derived from ATP. When conc of glucose high con of cAMP low so doesn't bind to CAP

No lactose - lac operon off

• lac repressor bound, blocking sigma70 of holoenzyme binding = no transctiption

Lactose and glucose - lac operon on but low transcription

• lac repressor bound
• Lactose changes repressor conformation = falls of operator site
• RNAP bind and transcribe but operon not efficient

Lactose only - lac operon on

• cAMP levels are high and bind to CAP
• CAP recognised by CTDs on a-subunits on RNAP
• CAP binds and causes conformational change
• Enhances binding and activity of RNAP = increased transcription

Riboswitch - regulatory segment of mRNA that binds a small mol in 5' untranslated region resulting in change in protein production

Most suppress gene expression - blocking termination/initiation of transcription

Examples - for adenine, lysine coenzyme B12, glycine

Torsional stress forms in front of RNA polymerase as it moves along dsDNA

Supercoil represses gene expression - prevents RNAP binding to DNA

Topoisomerases (ESSENTIAL) relieve and increase torsional stress = supercoiling/uncoiling

Uncoil DNA - nick 1 or both strands

Promote supercoiling - reseal broken strands

Two types:

Type 1 - single stranded cuts and connects 3' to 5' without ATP in eukaryotes

Type 2 - double stranded cuts and reconnects strands in ATP-dependent manner. Relieves both positive and negative supercoils