Fundamentals Scribe: Myra Dennis
Monday, September 14, 2009 (1st hour) Proof: Kosha Shah
Dr. Ryan Gene Expression – Prokaryotic and Eukaryotic Transcription Page 1 of 7
Continuing with prokaryotic gene transcription. We’ll focus on regulation in prokaryotes with grouping genes and operons. We’ll discuss 2 operons - the lac operon and the tryptophan operon. The second hour we’ll discuss eukaryotic gene expression and chromatin structure.
I. Gene Expression [S1]
II. Prokaryotic Chromosome (E. coli) [S2]
a. E. coli – has massive amount of DNA (4.6 million base pairs in one single, circular chromosome). The organism must find where its genes are, and which genes to transcribe at any particular time.
b. The major level of regulation in prokaryotes is at the transcription level. Identifying promotors, RNA polymerase, sigma factors, and also controlling which sigma factors are expressed. Grouping genes in operons is a way to control multiple genes at one time.
III. General Organization of Operons [S3]
a. General organization of an operon: It can have one or multiple structural genes, all driven off a single promotor.
b. Regulatory sequence called the operator that can be upstream, overlapping, or downstream of the promotor. Both the promotor and the operator region bind transcription factors, which are DNA binding proteins, which can increase or decrease transcription of this operon.
IV. Induction and Repression [S4]
a. Operons can be either induced or repressed.
i. If an operon is induced, transcription from that promotor is increased. The operons can be induced by metabolites. In this case, that metabolite is called a co-inducer. We’ll see an example of that with the lac operon.
ii. Decreased synthesis in response to a metabolite is termed repression, and the metabolite a co-repressor. We’ll see an example of that with the tryptophan operon.
b. Some substrates induce enzyme synthesis even though the enzymes can’t metabolize the substrate. This is an example of a gratuitous inducer. An example for the lac operon is IPTG, which we use in the lab a lot in order to turn on this promotor of the lac operon
V. Lac Operon [S5]
a. Schematic of the lac operon. The lac operon is regulated by positively and negatively.
b. DNA is 5’ to 3’. Here’s the +1 start site of transcription, and upstream of this the nucleotides are numbered negatively. Here’s the -10 box (TATA box we talked about), and the -35 box. There’s also an upstream sequence in the -61 region that can bind CRP, an activator protein that we’ll talk more about later.
c. Blue box - where RNA polymerase actually binds. The sigma factor recognizes the -10 / -35, the whole enzyme covers about 60 base pairs of DNA in the promotor, and it overlaps this repressor binding site. The lac operon is negatively regulated by a repressor that sits there. That blocks elongation of the RNA polymerase.
d. For most operons, multiple control mechanisms are the norm. So a lot of these have both positive and negative regulation.
VI. Lac Operon of E. coli [S6]
a. Here’s the promotor again with the operator.
b. Three structural genes: lacZ, lacY, and lacA. They encode for proteins.
i. LacZ – codes for the b-Galactosidase gene. It acts as a tetramer, so 4 of these come together for the active enzyme.
ii. LacY – encodes for a permease, which helps bring lactose into the cell.
iii. LacA – encodes for a trans-acetylase. People are still not quite sure what this does.
iv. When it can E. coli would rather metabolize glucose, because it doesn’t require any energy to bring glucose into the cell. But in the absence of glucose it needs to use other sugar substrates for metabolism. In the presence of lactose, lactose can actually induce this operon – it’s a co-inducer – to express the lacZ gene and the lacY gene. The lacY gene will help bring more lactose into the cell. The lacZ gene will help break lactose down into glucose and galactose.
v. Upstream of the promotor, there’s the lacI gene and a promotor which drives the expression of the lacI gene. It’s a constitutive promotor, so it’s always making lacI repressor, and lacI encodes for the repressor which binds to the operator region of the lac operon.
vi. If have mutation in this lacI gene, which blocks repressor binding, then all of these genes would be constitutively active, especially in the absence of glucose.
VII. The Lac Operon – Negative Regulation [S7]
a. The structural genes of the lac operon are controlled by negative regulation.
b. LacI gene product is the lac repressor. The lac repressor binds as a tetramer to the lac operator, and mutants in the lacI genes for lactose metabolism would be constitutively transcribed
c. The lac operator has palindromic DNA. We talked on Friday about how most of these transcription factor binding sites are palindromic in nature. It allows homodimers of transcription factors, in this case tetramer, to actually bind to that sequence.
d. The lac repressor – forms tetramer. The DNA binding portion of each of the monomers in this tetramer is in the N-terminal domain, and the inducer binding is in the C-terminal domain.
VIII. Lac Operon – LacI Gene Encodes a Repressor [S8]
a. Here’s the schematic: here’s the lacI gene, you make messenger RNA, it’s translated into repressor monomer, that forms a tetramer, and it binds to the operator. And even if RNA polymerase sits there it will block the elongation of it, so you don’t get transcription of the genes.
IX. Lac Operon – Negative Regulation [S9]
a. Operon is generally off, and it’s only fully on when lactose is present and glucose is absent.
b. When no lactose is present, the repressor is bound, inhibiting transcription of the genes.
c. When lactose is present, lactose is converted to allolactose, which binds to the repressor, causing it to fall off the operator and allowing transcription of the downstream cistrons.
X. Gratuitous Inducers [S10]
a. Here’s lactose, it has the b-Galactoside linkage. This is a substrate for b-Galactoside, it will cleave lactose, forming glucose and galactose.
b. I mentioned this gratuitous inducer earlier, IPTG. It has thio linkage, and it is not cleaved by B-Galactosidase. So it will bind the repressor, pull the repressor off the operator and you get constitutive expression of the genes.
XI. B-Galactosidase (LacZ) [S11]
a. The actual enzyme, LacZ, works as a tetramer and it will hydrolyze the galactosidic bond forming glucose and galactose.
XII. Lac Operator Sequence [S12]
a. It’s palindromic, so it reads similarly from 5’ to 3’ in both directions. It lies just downstream of the transcription start site, and it blocks transcription elongation.
XIII. Induction of the Lac Operon [S13]
a. How do you induce the lac operon? In the presence of this inducer, which is lactose, it binds the repressor, and keeps it from binding to the operator. It actually decreases the affinity for the operator sequence by about a thousand fold. You would get this polycistronic message made, which would be translated into 3 proteins.
XIV. Lac Operon: Catabolite Repression [S14]
a. Also regulated by catabolite repression, so E. coli can use several different substrates for its carbon source. It chooses glucose because it doesn’t need to expend energy to bring it into the cell.
b. It’s a glucose-sensitive operon – the expression of the operon is reduced in the presence of glucose.
c. Catabolism of glucose inhibits expression of these operons, called catabolite repression. There are numerous operons in E. coli that undergo catabolite repression. In the presence of glucose, it will block the transcription of this locus.
XV. Catabolite Activator Protein – Positive Control of the Lac Operon [S15]
a. You can positively activate the lac operon with the catabolite activator protein. It’s a transcription factor that promotes/increases the transcription through the locus. We showed its binding site is at the CAP site about -60, upstream of the transcription site, the +1 site. The CAP is a homodimer. Goes by 2 names:
i. CAP – Catabolite Activator Protein
ii. CRP – cAMP Receptor Protein
b. b. In the presence of cAMP, CAP will bind to the DNA, and form a closed promotor complex. So it increases the rate of formation of the closed promotor complex of the lac promotor.
XVI. Catabolite Activator Protein Mechanism of Activation [S16]
a. So how does this work? Normally, when you have plenty of glucose, half is inactive CAP. But in glucose starvation, you activate the enzyme (adenylyl cyclase) that will increase the cAMP levels in the cell.
b. cAMP will bind to the inactive CAP and make it active. Then it will bind to the upstream activation sequence there and turn on transcription of CAP controlled genes.
XVII. Catabolite Activator Protein [S17]
XVIII. So the lac operon can be positively regulated when CAP (shown here as a homodimer) binds cAMP when glucose levels are low, and it will bind to this upstream promotor sequence and actually bend the DNA.
XIX. CAP Activation of RNA Polymerase [S18]
a. This interaction is communicated to the RNA polymerase through the C-terminal domain of the alpha subunit of the RNA polymerase. In the activator site (about -60) and the promotor, the CAP cAMP dimer binds, interacts with the alpha subunit of RNA polymerase and increases the formation of the closed promotor complex.
XX. Dual Control of the Lactose Operon [S19]
a. What’s drawn here is the lac operon, and the lacZ gene, the operator, promotor, and the CAP site.
b. Four conditions:
i. Plenty of glucose, and no lactose, the E. coli would rather catabolize the glucose and in the absence of lactose, the repressor is bound to the operator and there is no transcription.
ii. In presence of lactose, and remove repression – if there’s plenty of glucose, you still don’t activate positively through CAP operon is still only expressed at very low levels.
iii. In the absence of glucose and lactose – both the repressor bound, blocking elongation because there’s not lactose to use. But in absence of glucose, do get CAP binding. Still no transcription because the repressor blocks it.
iv. No glucose, but lactose is present (bottom case) – only time the lac operon is fully transcribed. In this case, there’s activator protein binding, repressor is off, RNA polymerase comes in and you get full blown transcription.
XXI. The trp Operon – Co-Repressor Mediated Negative Control [S20]
a. An example of co-repressor mediated negative control. It encodes a leader sequence and 5 enzymes for tryptophan biosynthesis.
b. Trp operon is always on unless tryptophan levels in the cell are sufficiently high to turn it off. The cell doesn’t want to waste its time making more of these biosynthetic enzymes to synthesize more tryptophan if there’s tryptophan in the cell. It’s a way to shut down, repress trp operon when tryptophan levels are high in the cell.
c. In this case, the tryptophan is the co-repressor. If tryptophan levels build up, it binds to a repressor that then binds to the operator and blocks transcription.
d. Trp repressor binding excludes RNA polymerase from the promotor. It’s not blocking elongation here, it’s actually blocking binding to the promotor.
e. The trp repressor can also auto-regulate and it’s encoded by trp R gene. It auto-regulates it’s own expression.
XXII. Trp Operon [S21]
a. Here’s the trp operon. There are 5 structural genes (trpE, trpD, trpC, trpB, trpA). These encode for 3 enzymes involving changing chorismic acid to tryptophan.
b. The trpR gene is on another location on the chromosome, and that encodes for the repressor. There’s one polycistronic transcript. Preceding these 5 structural genes is the trpL sequence, the leader sequence. There are a number of biosynthetic operons for amino acids that have a similar type leader sequence.
XXIII. Regulation of the trp Operon by Repression and Attenuation [S22]
a. Trp operon is controlled by levels of Trp in the cell.
b. TrpR gene – encodes the repressor. With high levels of tryptophan, it will bind the inactive repressor, making it active and will bind the operator, blocking entry of the polymerase for the promotor.
c. Trp operon is also regulated by a transcription attenuation. In prokaryotes (absence of a nucleus), as soon as you transcribe messenger RNAs, you can start to have ribosomes line them and translate that message into protein. It’s different for eukaryotes. But in prokaryotes, you can have this coupled transcription/translation occurring.
d. Transcription attenuation is a way to modify transcript levels after transcription is already initiated. So you control the level of the 5 biosynthetic enzymes to synthesize tryptophan, after transcription is already started, the cell has a way of sensing the tryptophan levels. If tryptophan levels are high, then it makes an aborted transcript. The level of tryptophan is sensed during translation but the ribosome has to go through a couple of codons for tryptophan so it can sense the level tRNA that are charged with tryptophan. If there’s not much tryptophan around, the charged tRNA tryptophan levels will be low
e. So in high tryptophan levels, ribosome can speed through this leader sequence. There’s an intrinsic terminator sequence, and the transcript is terminated. When tryptophan is high, charged tRNAs for tryptophan are easily incorporated into ribosome, the ribosome blocks secondary structure formation, which causes anti termination, and you get termination
f. When tryptophan levels are low – ribosome stalls in this area without the charged tryptophan tRNA to incorporate into the 2 codons in the leader sequence. An alternative secondary structure forms, the anti terminator structure, and you get expression of the 5 structural genes.
XXIV. Attenuation [S23]
a. Attenuation is a way of sensing the need for tryptophan. It tests the need for tryptophan during translation of the leader sequence.
b. Depending on levels of charged tryptophan tRNA available to translate the codon UGG (there’s 2 of these tryptophan codons in the short leader sequence). If the ribosome translates these codons rapidly, transcription will stop because of an intrinsic terminator (there’s a G-C rich stem loop structure), and that’ll terminate the transcription at the end of the leader sequence. If the ribosome stalls on the trp codons, the RNA structure formed is not a terminator – transcription of trp operon proceeds.