4. Transcription and Regulation of Bacterial Gene Expression

Learning Outcomes: L9-10

• Familiarizing with the basic process of transcription
• Gaining a knowledge of the transcription machinery
• Understanding the steps of the transcription reaction
• Becoming aware of the role of s factors
• Gaining an understanding of transcription regulatory sequences (promoters, terminators)
• Becoming familiar with transcription termination mechanisms
• Familiarizing with the mechanisms of positive and negative regulation of gene expression
• Understanding the concept of ‘operon’
• Gaining a knowledge of cis-acting sequences
• Understanding the molecular mechanisms that underpin the regulation of the lac operon
• Appreciating the role of repressors and activators in regulation of gene expression
• Understanding how components of the lac system are used in biotechnological applications

Diapositiva 2

• Distinguish between basal and regulated transcription
• Know how RNA Polymerases II initiates transcription and know DNA elements and the factors required for transcription of genes
• Know that there are three RNA polymerases in eukaryotes RNA polymerase I, II, and III
• Understand that promoters are modular in nature and transcriptional activators have distinct DNA binding and transcriptional activation domains
• Know some of the methods used in studying transcription

Concept of Transcription

• Process of transcription= dsDNA transcribed to mRNA
• mRNA is identical to the coding strand and complementary to the template strand of DNA
• A transcription unit is a sequence of DNA transcribed into a single mRNA, starting at the promoter and ending at the terminator
• Can have single gene or multiple genes (operon)
• 

RNA Polymerase

• 
• The transcription bubble:
• Unwinding= RNA pol separates DNA strands to form a transient bubble to access template strand
• Add ribonucleosides to template in 5' to 3' direction (complementary base pairing)
• Hybrid of DNA and RNA produced (8-9bp)

RNA Polymerase

• RNA pol has 2 different nucleotide binding pockets
• 1= binds only purines- GTP and ATP, used for first nucleotide incorporated into the RNA transcript
• 2= binds any of the 4 ribonucleosides
• RNA has catalytic function of forming phosphodiester bond between the 2 nucleotides in the 2 binding pockets
• 3' OH mediates Nu attack on alpha phosphorus of second nucleotide to form bond and release 2 phosphates
• 

RNA Polymerase in Different Domains

• Has different complexities in different domains of life
• In each column= the subunits that make up polymerases
• Subunit number and complexity increases throughout domains
• 

RNA Polymerase in Different Domains

• Shape of polymerases (claw with channel) has been retained because all perform same function
• But have differences in their associated initiation and elongation factors when bound to DNA
• Yellow and blue= specific conserved motifs found in DNA at binding sites of RNA pol
• Pink subunit on bacteria= sigma factor
• ​​​​​​​Recognises and binds promoter on DNA
• Euk/archaea dont have subunit to recognise promoters and rely on other proteins to bind DNA promoter

RNA Polymerase in Different Domains

Bacterial RNA Polymerase

• Multi-subunit enzyme
• Contains channel/groove that could be region where DNA is accomodated
• Length of groove holds 16bp and groove is lined with positively charged aa's
• Exists in 2 forms: core and holoenzyme ​​​​​​​
• ​​​​​​​Holoenzyme contains extra subunit (sigma) to bind specifically at promoters
• Core can still bind stably to DNA but cant recognise promoter
• RNA pol is a good target for antibiotics
• 

Function of RNA Pol Subunits

Stages of Transcription

• Initiation:
• Includes all steps of transcription up to synthesis of first bond in RNA
• RNA pol finding promoter and binding
• Melting of DNA to form transcription bubble
• Elongation:
• RNA transcript extended by addition of ribonucleosides
• Elongation rate is alot slower than DNA polymerase- For DNA pol, polymerisation happens in few sites of genome, RNA pol can bind, transcribe and start again- one gene can be transcribed multiple times
• Termination:
• Ends transcription by stopping addition of ribonucleosides
• Causes dissociation of RNA pol from DNA and releases transcript​​​​

Initiation

• 
• 1. Holoenzyme binds DNA and forms closed binary complex (closed when DNA not yet denatured)
• 2. DNA melting, form transcription bubble= open binary complex
• 3. Uses template strand to incorporate ribonucleosides= ternary complex (RNA pol, RNA, DNA)
• Abortive initiation is common in bacteria
• 4. Promoter is cleared and sigma subunit may be released (not always)

Initiation: Abortive Initiation

• When RNA pol produces and releases a short transcript (8-10 nucleotides)
• But promoter has high affinity for RNA pol, RNA pol stuck and transcribes same section of DNA
• Eventually RNA pol extends one mRNA transcript and moves on downstream promoter- clears promoter to transcribe rest of genome

Promoters

• A sequence of DNA upstream a gene, key for transcription of that gene
• Promoter consensus sequence is constructed by aligning all known examples of bacterial promoters and defining which is the predominant base at each position
• Conserved features of bacterial promoters are:
• Start point- usually a purine
• -10 TATA box= A 6bp motif centred around the -10
• -35 box= A 6bp sequence centred about the -35
• Some promoters contain A-T rich sequence upstream of -35 = UP element (contacted by alpha subunit)

Promoters

Example Promoters Recognised by E.coli

• 
• Changing one conserved base can change the activity of the promoter
• Either up or down regulated

Sigma Factors

• 
• Directly contact the DNA
• Functions:
• Promoter recognition
• Promoter melting
• Decreasing affinity of RNA pol holoenzyme for random sequences of DNA
• Sigma 1.1= involved in autoinhibition
• Sigma 2 and 3 involved in binding -10 box
• Have trp, tyr, phe aa's which are involved in denaturing and opening up DNA
• Sigma 4 makes contact with -35 box

Sigma Factors: Elongated Shape

• 
• Bound to RNA pol, sigma factor is spread over the surface
• When free in cytosol, is not able to bind DNA due to autoinhibition (allosteric control)
• N- region binds and occludes DNA binding domains in C-
• ​​​​​​​Upon binding, N- swings over 20A and unblocks domains

Alternative Sigma Factors

• Each bacterial cell contains more than one type of sigma factor
• Each specific for transcribing a different set of genes
• σ70 is the primary (housekeeping) sigma factor in E.coli
• Transcribes most genes
• In emergencies specialised sigma factors replace housekeeping
• eg σ32 heat shock factor- when exposed to high T E.coli needs specialised proteins
• In Bacillus subtilis a cascade of sigma factors regulate sporulation
• 

Termination

• RNA polymerase moves along the DNA template until it meets a terminator sequence
• 2 different types of termination mechanisms in bacteria: Rho-dependent and Rho-independent
• 

Termination: Rho-Independent

• 
• Intrinsic terminator sequence possess:
• 2 symmetrical GC rich segments
• Downstream run of 4-8 A residues
• When RNA transcribes the GC rich regions it pauses- more energy needs to break the 3 bonds
• Allows time for transcript to form stem and loop structure (GC regions= stem)
• RNA pol transcribes downstream A's, RNA pol falls apart due to steric hindrance in the ternary complex due to stem and loop
• Releases transcript

Termination: Rho-dependent

• Requires:
• Rho protein, hexameric helicase and ATPase
• Rut site sequence- sequence on RNA transcript recognised by Rho
• Possible auxillary sequences
• Rho protein structure:
• ATPase activity in each subunit in C- domain
• N-terminal domain (green) is responsible for binding RNA
• Start of RNA transcript (5') bound to internal site
• Rho is a gapped ring before binding RNA transcript, closed ring upon binding
• 

Termination: Rho-dependent

• 
• 1. RNA pol transcribes DNA
• 2. Rho binds Rut on RNA transcript
• 3. Using ATPase activity rho translocates on the RNA transcript until reaching RNA pol
• Unwinds the DNA/RNA transcript and release transcript
• RNA pol falls apart and ternary complex disassembled

Transcriptional Regulation Terms

• Cis-acting sequences- Sequences on DNA that only affect DNA regions attached to them (cis), eg promoter/terminator, do not encode gene product
• Trans-acting factors/products- Proteins that move away from the site of synthesis and act anywhere in the cell
• Repressor- Protein that binds sequences on DNA and prevent transcription, block RNA pol accessing the promoter
• Operator- cis sequence on gene to which repressor binds
• Transcription factor- proteins that directly interact with RNA pol and help start transcription​

Basics of Regulating Transcription

• Negative regulation:
• A trans-acting repressor binds to the cis-acting operator to turn off transcription
• RNA pol cannot access promoter
• Operators always downstream of promoters
• 

Basics of Regulating Transcription

• 
• Positive Regulation:
• Trans-acting factors must bind to cis-acting sites in order for RNA pol to initiate transcription at the promoter
• One or several proteins

The Lac Operon

• Genes coding for proteins that function in the same pathway may be located adjacent to one another and controlled as a single transcription unit, transcribed as a single polycistronic mRNA:
• Co-regulation of these genes
• The lac operon includes cis-acting elements and protein-coding genes
• ​​​​​​​LacZ = B-galactosidase, cleaves lactose into glucose and galactose
• LacY = permease membrane protein responsible for uptake og lactose into bacterial cells
• LacA = transacetylase, transfers acyl group from acetyl coA onto B-galactosides (sugars)
• Upstream is an independent transcriptional unit containing promoter and lacI gene that encodes the repressor of the lac operon​​​​​​​

The Lac Operon

Expression of Lac Genes

• 
• Expression controlled by repressor protein encoded by lacI gene
• Repressor binds operator, operator overlaps the promoter
• In the absence of lactose bacteria dont need to make enzymes to uptake and metabolise lactose:
• Repressor binds operator
• RNA pol cant access promoter

Expression of Lac Operon can be Induced

• Induction= synthesis of enzymes in response to appearance of a specific substrate in the cell
• Small molecules that induce the operon are identical to or related to the substrate for its enzymes (gene products)
• B-galactosidases are substrates for the enzymes encoded by lacZYA genes (eg lactose, IPTG)
• Absence of B-galactosidases= lac operon expressed at low (basal) level
• Presence of specific B-galactosidases= induces transcription of all 3 genes

Expression of Lac Operon can be Induced

• When lactose/IPTG (inducers) bind repressor, repressor undergoes conformational change and can't bind the operator
• Repressor (tetramer) contains binding site for the DNA operator and for the inducer
• Binding of one side effects the binding on the other side= allosteric control
• 

Identifying Operator: Cis-acting Mutations

• Cis-acting constitutive mutations in the operator caused constitutive expression of all 3 lac genes
• Mutation in operator= repressor cant recognise or bind operator
• RNA pol can bind promoter
• Constitutive= lac operon always transcribed whether lactose present or not because repressor can never bind operator
• Mutations are cis-acting and only affect those genes on the contiguous stretch of DNA (adjacent to the operator) - ONLY AFFECT THAT SPECIFIC OPERATOR

Identify Repressor:Trans-acting Mutations

• Trans-acting mutations in the lacI gene:
• Effect expression of all lacZYA clusters in bacterium
• Mutation in lacI gene on chromosome, leads to production of defective repressor that wont bind operator where lactose present or not
• Constitutive expression of lac operon
• Partial diploidy of Lac genes- in plasmid as well as on chromosome:
• When there are no mutations lacI repressor will repress transcription of lac genes in both plasmid and chromosome
• In a situation of diploidy the WT (non mutant) lacI gene would win over the mutant (non-functional gene) so mutants are reccessive

LacI Repressor Structure

• 
• Repressor monomer contains several domains:
• N-terminal DNA binding domain: contain 2 short α-helical regions that bind major groove of DNA
• Hinge: connects DNA binding domain to core
• Core domain: inducer binding site in a pocket between core subdomains 1 and 2
• Helix at C-terminal: Involved in oligomerisation (assembly of dimers/tetramers)

How Repressor Binds DNA

• The DNA binding domain of each monomer (helix) within a dimer inserts into the adjacent major groove of DNA
• The hinge helix inserts into the minor groove of operator DNA
• 

Allosteric Control Regulates DNA Binding

• 
• Active Repressor Conformation:
• The 2 DNA binding domains of a dimer can insert into successive turns of the helix
• Inducer binding:
• Disrupts hinge= change conformation
• 2 DNA binding sites not in right geometry to make simultaneous contact with the DNA

Operator Symmetrical Sequence

• 
• Repressor binds dsDNA sequence on operator
• The operator consists of 2 inverted repeats= palindromic sequence of 26bp
• Each monomer of repressor contacts one half site
• Palindromic sequence= reads the same on each strand of DNA when read in 5' to 3' direction

Lac Operon has More than One Operator

• 
• Full repression requires the repressor to simultaneously bind:
• O1- operator at lacZ promoter
• Additional operator downstream or upstream
• Each dimer in the repressor binds one operator causing DNA looping which loops around region of promoter and turns off expression of lac genes

Crystal Structure of Repressor Bound

• 
• Protein X is CRP activator which binds to target sequence adjacent to the promoter region

Positive Regulation of Lac Operon

• Mediated by CAP(or CRP) activator protein that binds to a target sequence (cis-site) adjacent to the promoter
• CAP dimer is activated by single molecule of cAMP
• When active it binds target sequence upstream promoter and introduces a 90 degree bend into DNA at its binding site
• CRP makes physical contact with RNA pol and assists transcription of lac genes

Catabolite Repression

• cAMP produced in cell from ATP by Adenylate cyclase
• Inverse correlation between glucose and cAMP
• Glucose inhibits production of AC
• High Glu= low cAMP
• 

Biotechnology Applications of Lac Operon

• Expression Vectors: the T7 expression system
• E.coli bacteria host that contains:
• Host genome with integrated segment of DNA (bacteriophage)
• DE3 bacteriophage contains stretch of DNA with P lac promoter, operator and T7 RNA pol (polymerase of T7 phage and is a single polypeptide not multisubunit)
• Within host genome is also LacI repressor
• Expression vector with target gene is transformed into host
• Target gene is expressed under control of T7 promoter which is recognised by specific T7 RNA pol

T7 Expression System

T7 Expression System

• Absence of IPTG/lactose:
• Repressor binds operator and prevents transcription of T7 RNA pol
• Presence of IPTG/lactose:
• ​​​​​​​Repressor becomes inactive
• E.coli RNA pol will access the lac promoter and transcribe gene encoding T7 RNA pol
• T7 RNA pol binds T7 promoter in expression plasmid and abundantly transcribes target