MICB 211: chapter 8, gene expression and regulation

promoter facts

• sequence of DNA where RNA pol binds to

• RNA pol binding facilitates transcription

• Sigma factor in the RNA pol recognises the promoter and initiates the binding

RNA pol facts

• enzyme in transcription that synthesizes RNA from DNA.

• it binds onto the promoter and facilitates transcription

• It first reads the DNA strand 3’ → 5’, unwinds the DNA, forming a transcription bubble.

• Then the RNA strand is synthesized from 5’ to 3’

RNA pol holoemzyme

2 alpha subunits + 2 beta subunits + sigma factor

sigma factor/ subunit

• protein in middle of the RNA pol holoenzyme (bacteria)

• recognises promoters based on their DNA sequence (consensus sequence).

• RNA pol can weakly bind to any part of the DNA non-specifically, and the sigma factor can lead it to the promoter region, then transcription can begin.

• There are a lot of different sigma factors in bacteria that identify different sets of promoters and specify different promoter affinities.

• One sigma factor can control multiple genes, can bind to several different promoters and can regulate multiple genes at once.

consensus sequence

• sequence on promoter that sigma factor finds

• sequence with the highest binding affinity for the sigma factor

• Any deviation from the consensus is bound less strongly and less frequently

• well- conserved nucleotide sequence, then N(number), then more nucleotide sequence

Promoter affinities

the binding strength for different promoters

operator

• short section of DNA

• found before/after/overlapping the promoter.

• Location of the operator depends on the specific gene or operon that it regulates. 

• The binding site for regulatory proteins, either (repressors/inhibitor proteins or activator/ promotion proteins.)

• They regulate/control the expression of genes, if they are expressed or not, and how strongly they bind.

• operator section of DNA does not code for any proteins itself, the repressor/ activator proteins bind onto it.

• Operators are mostly found in prokaryotic organisms.

coding region

• the section of DNA (gene and exerts activity) that is used as a template for RNA synthesis.

• A protein is then usually made from the synthesized RNA.

• The RNA pol transcribes the DNA into RNA moving from 3’ → 5’. The RNA is made from 5’ → 3’.

• In prokaryotes the RNA is called mRNA, and in eukaryotes a 5’ cap and a poly-A tail is added.

• Then ribosomes synthesize the protein from RNA.

• The coding region is also a functional component because it is the coding for the specific sequence of RNA, and the RNA codons give function to the final gene product. Some products might be (protein, tRNA, rRNA)

operons

• contains multiple genes of DNA controlled by a single promoter.

• The genes all share the same promoter, (operator) and terminator.

• in transcription there is 1 RNA binding event.

• Since multiple genes can be in one RNA, multiple proteins can also be transcribed.

• Having multiple genes in an operon is more efficient and saves time and energy.

• This is effective in genes involved in the same process (lac operon), it has different genes that overall helps in transcription regulation.

• Another way of being effective are forming protein complexes, multiple genes can together make an important amino acid, and the genes/ proteins function together.

How is gene transcription controlled?

• Single genes of operon expression: Important so genes are expressed at the right time in the right place. 

• By the molecule that the operon is dependent on (SIGNAL MOLECULE). This is called the genetic regulation of a signal molecule that is in the genetic level of regulation

regulatory proteins

bind to operator

repressor proteins

• bind to operator (downstream)

• block RNA pol binding to P

• -ve regulation

activator proteins

• bind to operator (upstream)

• help RNA pol bind to P

• +ve regulation

+ve regulation (activator present vs absent)

Activator is present: 

• Activator protein binds to operator

• Recruits RNA pol

• Transcription happens

Activator is absent:

• Nothing binds to operator

• RNA pol is not recruited

• Transcription does not happen/ happens in small amounts

-ve regulation (repressor present vs absent)

Repressor is present: 

• Repressor protein binds to operator

• RNA pol is blocked from binding

• Transcription does NOT happen

Repressor is absent:

• Nothing binds to operator

• RNA pol can bind to the promoter

• Transcription does happen

operons

contain the promoter, operator, coding genes, and coordinate expression of genes. They include operators.

directionality of RNA pol moving on DNA template

3’ --->5’

directionality of mRNA synthesis

Ribosomal binding site/ start codon 5’ →3’

ribosome directionality of RNA

5’ →3’

directionality of polypeptide chain synthesis

N(met, comes out FIRST, ) →C(closest to mRNA strand)

gene regulation/ expression: what, when, where, why, how much

what: which genes are turned on or off

when: control timing of gene expression, development, cell cycle, responding to signals

where: specific tissues or cell types

why: Limited resources and conserving energy. Cell specialization, different cells perform different functions even though they all contain the same DNA. all necessary genes are active in each cell type There is no point in making proteins you don't need.

how much: depends on signal molecule

what are 4 ways bacteria control expression of genes

1. Promoter strength

2. Regulatory proteins

3. mRNA stability/ degradation

4. Protein degradation

promoter strength control

• Control gene expression at DNA/ transcriptional level

• Determines: how much mRNA is produced, binding of RNA pol, amount of expression

• The most useful technique bc it prevents the synthesis of proteins from the start

• Strong promoter: stronger binding/ association  with RNA pol, more frequent gene expression of the gene

• Weak promoter: weak binding with RNA pol, less frequent gene expression

Regulatory proteins control

• Control gene expression at DNA/ transcriptional level

• Determines: how much mRNA is produced

allostery/ allosteric regulation

• mechanism that when a molecule (effector/modulator) binds at one site of a protein/ enzyme, the protein/enzyme go through a conformational change at the functional site and it regulates its activity

• interactions with small molecules (metabolites) onto regulator protein results in a switch confirmation of the protein.

arg operon facts

• Arginine: a small signal molecule (effector/metabolite) that regulates ArgR protein activity

• ArgR: a protein that regulates the Arg operon. Typically a repressor protein that inhibits transcription of the arg operon when bound to the effector molecule It is a DNA-binding repressor protein. This protein is constitutively expressed, as made and with the argR gene.

• Arg operon: the synthesis of arginine amino acid.

ArgR activator, low arginine levels

• +ve regulation

• Function of operon is to create more arginine molecules. 

• activator protein (ArgR) = Operon is upstream of the P

• ArgR protein is considered is an enhancer 

• ArgR protein will not bind to arginine molecules -- as there is not much of the molecule -- ArgR protein will instead bind to the DNA operator. So the arg operator is unblocked and there is no repression of the operon

• Since ArgR is an activator/ enhancer protein, it recruits RNA pol and RNA pol is able to bind and transcribe Arg operon genes.

• There are high levels of transcription and it produced arginine

• Arginine molecule is considered an inhibitor because it inhibits ArgR from doing its job. Its considered an effector that affects/ regulates the activator activity

• Produces: arginine

ArgR activator, high arginine levels

• -ve regulation

• Arginine is present in high concentrations, you don't want to make more of the molecule

• ArgR protein binds to arginine molecule as a corepressor and undergoes a conformational change called now the ArgR-arginine complex

• ArgR protein is considered is an effector and regulates activator/ repressor activity

• This activated complex binds to DNA operator preventing/ blocking RNA pol from binding

• Operon is not turned on and can not transcribe Arg operon genes. No arg operon expression

• Nothing is produced and E is conserved

• Arginine molecule is considered an inhibitor because it inhibits ArgR from doing its job

• Produces: NOTHING, E is conserved

ArgR repressor, low arginine levels

• Operon is found after promoter

• Don't want ArgR to bind to the DNA because if ArgR would repress expression of the arg operon. There are low levels of arginine, needs to make more of the molecule so needs expression

• ArgR protein does not bind to DNA operator at low concentrations of arginine signal molecules. This is because to act as a repressor ArgR would be called a corepressor, which consists of ArgR + arginine. It cannot corepress.

• RNA pol can bind onto operator since ArgR cannot

• Transcription happens if the arg operon to produce arginine molecule

• Arg are represses arginine expression 

Arginine is an activator molecule

ArgR repressor, high arginine levels

• ArgR protein binds to arginine and acts as a corepressor. This corepressor can now bind to the operator and repress expression of the arg operon!

• RNA pol can NOT bind to the DNA, the repressor is in the way!!

• ArgR is a repressor protein

• Arginine is an activator of argR

mRNA stability/ degradation control

• Control gene expression at translational level

• Determines: how much protein is produced

Diauxic growth curves

y axis: OD

x axis: time

1. lag of first C source

2. log of first C source (primary source taken up)

3. lag of second C source

4. log of second C source (seconday source taken up)

5. stationary phase

6. death phase

why is glucose the preferred C source

• produces the most energy per unit time

• Glucose actually has regulatory systems that block out the intake of other sugars

carbon catabolic repression (CCR) pathway

• Molecular mechanism that represses lactose consumption.

• This pathway has a preference towards glucose and inhibits the metabolism of sugars that are not glucose (like lactose).

• In an environment with both glucose and lactose, glucose will be used up first, then lactose. When glucose is detected, the transport of other sugars are not allowed as well

• regulation at gene level (inhibiting transporter proteins/ lac operon)

lac operon: glucose

• +ve regulation (recall// binding of protein enhances)

• cAMP: small molecule that is present when glucose is not. (inverse relationship)

• CAP: activator protein, cAMP dependent, activator of the lac operon, recruiting RNA pol to DNA

• CAP site: where cAMP and CAP bind when cAMP and CAP bind together first. it enhances CAP site and transcription

lac operon: lactose

• -ve regulation (recall// binding of protein restricts)

• Lactose: signal molecule

• LacI: regulatory protein, repressor protein  that binds to operator in order to block RNA pol

lac operon: No G, yes L

(level) High lac operon expression: CAP site enhances the transcription and  lactose allows for the transcription to happen

lac operon: high G, no L

(level) no expression of the lac operon

lac operon: no G, no L

(level) no expression of the lac operon

lac operon: high G, high L

(level) weak/basal expression

Protein degradation control

• Control downstream impact of gene presence

• prevents overaccumulation, signals

• Determines: how much protein is around to do its job

• Worst option you use so much energy

mRNA degredation

• unstable form of RNA

• degraded by RNAses

• RNAses recognise poly-A tail on mRNA to break down

• some RNAses break up untagged/ tagged mRNA

proteases

• enzyme that uses an inducer to degrade bacterial protein.

• This process is very energy- intensive because.

• Some bacteria also have a proteasome-like system found in eukaryotes. T

• he inducer binds to the tag on the protein. The inducer helps the tag (and protein) get eaten up by the ClpX/P protease

1. Proteasome-like system:  a multi- protein complex in bacteria and unicellular organisms. 

HGT

• Obtaining genes from within the same generation

• Gene donor is non-parent

• Organism receiving gene is the recipient

• It allows bacteria to acquire new genes and evolutionize by changing their genotype.

• Changing the genotype affects their phenotype without reproducing OR slowing the process of evolution.

  • Implications in evolution, antibiotic resistance, and more

types of HGT

1. transformation

2. transduction

3. conjugation

types of HGT: transformation

• transported naked linear DNA from lysed microbe into the host

• recipient cells must be in a competent state to accept DNA

competent state for cells

• chemically or electrically treated to allow them to take up foreign DNA

• treated with chemicals, then heat shock

• electric shock

types of HGT: transduction

• uses bacteriophage to transport plasmid DNA into cell

• transfer of DNA from a donor → recipient by a virus (phage)

• Virus can accidentally package parts of the host's DNA when going through a lytic cycle. Phage with only host DNA= transducing phage

types of HGT: conjugation

• when 2 prokaryotic cells transfer a plasmid when they directly conduct

How does it work?(steps)

1. Conjugative plasmids encode transfer proteins

2. Pilli allow contact between two cells

3. Pili retracts