Regulation of Metabolic Pathways

Events at the DNA replication fork

Helicase unwinds the helix, single-strand binding proteins stabilize the strands, topoisomerase/gyrase relieves supercoiling ahead of the fork, primase makes RNA primers, and DNA polymerase III adds nucleotides 5' to 3' (continuous on the leading strand, Okazaki fragments on the lagging strand). DNA pol I replaces primers with DNA and ligase seals nicks.

How does one DNA strand's sequence determine the complementary strand and the mRNA?

Base pairing rules (A-T, G-C in DNA; A-U in RNA) set the sequence. Template 3'-TAC-5' gives complementary DNA 5'-ATG-3' and mRNA 5'-AUG-3'. The mRNA is complementary to the template strand and matches the coding strand, with U in place of T.

What does it mean that the two DNA strands are complementary?

They are antiparallel (one 5'->3', the other 3'->5') and held together by hydrogen bonds between bases A=T (2 bonds) and G≡C (3 bonds), so the sequence of one strand determines the sequence of the other.

Advantages of metabolic regulation using the central dogma

Cells can control expression at several levels (transcription, translation, and enzyme activity) so they make only the enzymes they need. This saves energy, ATP, and amino acids, lets them respond quickly to the environment, and coordinates pathways.

Rho-dependent vs rho-independent termination of transcription

Rho-independent (intrinsic): the mRNA forms a GC-rich hairpin followed by a run of U's; the hairpin stalls RNA polymerase and the weak U-A pairs let the RNA dissociate. Rho-dependent: Rho protein (helicase, uses ATP) binds the rut site on mRNA, chases RNA polymerase, and unwinds the RNA-DNA hybrid when it pauses, releasing the transcript.

Generic structure of a bacterial operon (diagram)

5' [Activator binding site] - [Promoter] - [Operator] - [Structural gene 1] - [gene 2] - [gene 3] - [Terminator] 3'. A regulatory gene elsewhere encodes a repressor or activator. Activator site: activator binds and helps RNA polymerase. Promoter: RNA polymerase binds. Operator: repressor binds and blocks RNA polymerase. Structural genes: encode the enzymes.

Activator binding site

DNA sequence (upstream of the promoter) where an activator protein (e.g., CAP-cAMP, MalT) binds to help RNA polymerase bind the promoter and increase transcription (positive control).

Promoter

DNA sequence where RNA polymerase (with sigma factor) binds to begin transcription of the operon.

Operator

DNA sequence next to or overlapping the promoter where a repressor protein binds and blocks RNA polymerase (negative control).

Structural genes

The genes in an operon that code for the proteins (usually enzymes of one pathway); transcribed together into one polycistronic mRNA.

How does a regulon function to control expression of multiple operons?

A regulon is a set of operons (often at different places in the genome) controlled by the same regulatory protein or signal. One regulator (e.g., CAP-cAMP) turns all of them on or off together so the cell responds to one condition in a coordinated way.

Relationship between genes, mRNA transcripts, and proteins from one operon

One promoter makes one polycistronic mRNA that carries all the genes; each gene has its own ribosome binding site and start/stop codons, so each is translated into a separate polypeptide. n genes -> 1 mRNA -> n different proteins (many copies of each).

Two genetic elements that make up a prokaryotic genome

The chromosome (essential genes) and plasmids (extrachromosomal, usually nonessential, DNA).

Advantages of coupling transcription and translation for metabolic adjustment

With no nucleus, ribosomes start translating the mRNA while it is still being made. Cells can change protein levels within minutes, and shutting off transcription quickly stops protein production. This fast response saves energy and lets bacteria adjust to environmental change.

Why does one operon mRNA have multiple ribosomes bound?

Many ribosomes translate the same mRNA at once (a polysome), so each transcript makes many proteins quickly (amplification), and each ribosome can start at the different genes of the polycistronic mRNA. mRNA is short-lived, so this maximizes protein made per transcript.

Advantages of organizing genes in operons

Genes for a single pathway are controlled by one promoter/operator, so they are turned on and off together and made in equal amounts at the same time. Requires fewer regulatory elements and saves energy.

Benefit of gene expression influenced by external (environmental) and internal (cellular) molecules

External molecules (nutrients such as lactose or maltose) tell the cell what is available; internal molecules (end products such as arginine or tryptophan) tell the cell what it already has. Together they ensure enzymes are made only when needed, conserving energy and boosting fitness.

Determine operon regulation from a growth curve

Compare growth of wild-type and regulatory mutants with and without the signal molecule. If the sugar is the only carbon source, growth means the operon is expressed. Example: a repressor (lacI) mutant grows on lactose even without inducer/constitutively, suggesting negative control; an activator mutant fails to grow even with inducer, suggesting positive control.

Determine operon regulation from cell viability data

Count viable cells (CFU) under each condition. Loss of viability or no colonies when a needed pathway can't be expressed (e.g., an anabolic operon with a mutation on media lacking the end product, or a catabolic operon mutation with the sugar as the sole carbon source) shows the operon is required; compare mutants to the wild-type to see which regulator or site is affected.

Determine operon regulation from gene (mRNA) expression data

Measure operon mRNA (RT-qPCR, Northern blot, RNA-seq) under each condition. More mRNA with the molecule present = induction (inducer); less mRNA with the molecule = repression (co-repressor). Constitutive mRNA in a regulator mutant shows what that regulator normally does.

Determine operon regulation from protein-binding-DNA data

Gel shift (EMSA), footprinting, or ChIP show which protein binds which DNA site and when. Repressor bound to the operator only with co-repressor = repressible. Repressor bound to the operator only without inducer = inducible. Activator bound to its site only with inducer = positive control.

Diagram: Arginine operon with and without arginine

Without arginine: ArgR repressor is inactive, cannot bind the operator; RNA polymerase transcribes argA-E; arginine-making enzymes are produced (operon ON). With arginine: arginine acts as co-repressor and binds the repressor; the active repressor binds the operator and blocks RNA polymerase; no transcription (operon OFF). Negative control, repressible.

Diagram: Lactose operon in -Lac/-Glu

No lactose: the lac repressor (LacI) is active and bound to the operator. Low glucose means high cAMP, so CAP-cAMP binds the activator site, but the repressor still blocks RNA polymerase. Operon OFF.

Diagram: Lactose operon in -Lac/+Glu

No lactose: repressor bound to operator. High glucose means low cAMP, so CAP is not activated and not bound. Operon OFF.

Diagram: Lactose operon in +Lac/-Glu

Lactose (allolactose, the inducer) binds the repressor so it cannot bind the operator. Low glucose means high cAMP, so CAP-cAMP binds the activator site and helps RNA polymerase bind the promoter. Operon fully ON (maximal transcription of lacZYA).

Diagram: Lactose operon in +Lac/+Glu

Allolactose inactivates the repressor (operator free), but high glucose means low cAMP so CAP-cAMP doesn't bind. RNA polymerase binds weakly, so only low-level (basal) transcription. Catabolite repression.

Diagram: Maltose operon with and without maltose

Without maltose: the activator (MalT) is inactive and cannot bind the activator site; RNA polymerase binds the promoter poorly; operon OFF. With maltose: maltose (inducer) binds MalT, the active activator binds the activator site and helps RNA polymerase bind the promoter; mal genes are transcribed (operon ON). Positive control, inducible.

Compare and contrast regulation of the arginine, tryptophan, lactose, and maltose operons

Arginine and Tryptophan: anabolic, repressible, negative control; repressor is inactive until the end product (arg or trp = co-repressor) binds it; trp also uses attenuation. Lactose: catabolic, inducible; negative control by LacI (inactivated by allolactose) plus positive control by CAP-cAMP (catabolite repression). Maltose: catabolic, inducible, positive control by the MalT activator (needs maltose).

Determine negative vs positive control from cell viability or growth curve data

Negative control: a mutation inactivating the regulator (or the operator) causes constitutive expression, so growth/expression occurs even without the inducer. Positive control: a mutation inactivating the activator (or its site) prevents expression, so no growth even when the inducer is present.

Why are anabolic pathways typically negatively controlled (repressible)? Arginine operon model

The cell needs the end product (arginine) when none is available, so the default state is ON (repressor inactive). When arginine is plentiful, it acts as a co-repressor, activates the repressor, and shuts the operon OFF, saving energy and materials. Feedback by the end product.

Why are catabolic pathways typically positively controlled (inducible)? Maltose operon model

The enzymes are only useful when the substrate (maltose) is present, so the default is OFF. Maltose (inducer) activates the activator to turn the operon ON, so no energy is wasted making enzymes for a sugar that isn't there. Positive control also allows preferred sugars to override it.

Compare the repressor of the arginine operon vs the lactose operon

Both bind the operator and block RNA polymerase. Arginine repressor: inactive by itself; needs co-repressor (arginine) to bind the operator (repressible). Lac repressor: active by itself and bound to the operator; the inducer (allolactose) binds it and releases it from the operator (inducible).

Compare inducers and co-repressors

Inducers (allolactose for lac, maltose for mal) turn the operon ON: allolactose binds and inactivates the lac repressor; maltose binds and activates MalT. Co-repressors (arginine, tryptophan) turn the operon OFF by binding the inactive repressor so it can bind the operator. Inducers are usually substrates of catabolic pathways; co-repressors are end products of anabolic pathways.

Explain diauxic growth of E. coli in glucose + lactose (repressor, activator, catabolite repression)

Phase 1: glucose is present, so cAMP is low and CAP is inactive; allolactose may release the repressor, but without CAP there is only basal lac transcription, so cells use glucose. When glucose runs out, cAMP rises, CAP-cAMP binds the activator site, and with allolactose inactivating the repressor the lac operon turns fully ON. There is a lag while lac enzymes are made, then a second growth phase on lactose.

Why do cells use post-transcriptional regulation of metabolic pathways (central dogma)?

After transcription, mRNA and proteins already exist, so regulating translation or enzyme activity gives a faster, finer, reversible response than changing transcription. It lets cells immediately stop wasting energy on making unneeded product and fine-tune levels.

Small RNA (sRNA) regulation

Non-coding RNA that base-pairs with a complementary target mRNA (often with the Hfq chaperone). Usually blocks the ribosome binding site (stops translation) and/or promotes mRNA degradation by RNases (sometimes opens a hairpin to activate translation). Acts at the mRNA level; fast and reversible.

Riboswitches

Part of the 5' end of the mRNA itself. The aptamer region binds a specific small molecule (a metabolite) directly and changes the mRNA shape, either hiding the ribosome binding site (stops translation) or forming a terminator (stops transcription). Needs no protein.

Allosteric regulation / feedback inhibition of enzymes

The end product binds an allosteric site on an enzyme (often the first/committed step), changing its shape so the active site is inactive. Acts on existing protein, so it is immediate and reversible, with no change to gene expression.

Compare sRNA, riboswitches, and feedback inhibition (diagram)

sRNA: separate RNA binds target mRNA -> blocks translation/degrades mRNA. Riboswitch: the mRNA binds the metabolite directly -> shape change blocks translation/transcription. Feedback inhibition: end-product binds the enzyme protein -> enzyme inactivated. Levels: sRNA/riboswitch = mRNA (post-transcriptional); feedback = protein activity. Speed: feedback is fastest.

Tryptophan operon: regulation (with and without tryptophan)

Without tryptophan: the Trp repressor is inactive and can't bind the operator, so RNA polymerase transcribes trpEDCBA and tryptophan is made (ON). With tryptophan: tryptophan is the co-repressor, binds the repressor, the active repressor binds the operator and blocks transcription (OFF). Negative control, repressible. The trp operon also has attenuation: when tryptophan is plentiful, the ribosome moves quickly through the leader peptide, the mRNA forms a terminator hairpin, and transcription stops early.

Inducer or co-repressor of the arginine operon

Arginine is the co-repressor. It binds the inactive ArgR repressor, which then binds the operator and shuts the operon OFF.

Inducer or co-repressor of the tryptophan operon

Tryptophan is the co-repressor. It binds the inactive Trp repressor so it can bind the operator and turn the operon OFF (it also promotes attenuation).

Inducer or co-repressor of the lactose operon

Allolactose (made from lactose) is the inducer. It binds the LacI repressor, which releases the operator and turns the operon ON. cAMP also acts as a signal: it binds CAP, and CAP-cAMP activates transcription when glucose is low.

Inducer or co-repressor of the maltose operon

Maltose is the inducer. It binds the MalT activator, which then binds the activator site and turns the operon ON. No co-repressor.