FCH 532 Lecture 16

Extra credit assignment for Friday March 2-Seminar Speaker Matt DeLisa, 3PM-4:30PM 148 Baker

New Homework/Study Guide postedChapter 31

Major classes of RNA

• Ribosomal RNA (rRNA)

• Transfer RNA (tRNA)

• Messenger RNA (mRNA)

• The role that proteins are specified by mRNA and synthesized on ribosomes was defined by experiments in enzyme induction.

• Enzyme induction is a consequence of mRNA synthesis by proteins that specfically bind to the mRNA’s DNA template.

Enzymes• Constitutive enzymes - enzymes that are involved in

basic cellular housekeeping functions and are synthesized at a constant rate.

• Adaptive/inducible enzymes-enzymes that are synthesized at rates that vary with the cell’s circumstances.

• Example: lactose metabolizing enzymes conversion of lactose to galactose and D-glucose.

• Two main proteins: -galactosidase-catalyzes the hydrolysis of lactose to galactose and glucose

• Galactosidde permease (lactose permease)-transports lactose into the cell.

Enzymes

• In the absence of lactose only a few molecules of -galactosidase or lactose permease.

• Minutes after lactose introduced increased by ~1000-fold.

• Lactose unavailable - returns to the basal level.

• Metabolic product triggers the synthesis of proteins-inducer.

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Figure 31-1 The induction kinetics of -galactosidase in E. coli.

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Inducers of the lac operon

• Physiological inducer-lactose isomer 1,6-allolactose.

• Results from transglycosylation of lactoase by -galactosidase

• Most studies of lactose system use isopropylthiogalactoside (IPTG).

• Potent inducer that is not degraded.

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Figure 31-2 Genetic map of the E. coli lac operon.

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lac repressor inhibits synthesis of lac operon proteins

• PaJaMo experiment-Hfr bacteria of genotype I+Z+ were mated to F- strain with a genotype I-Z- in the absence of inducer while -galactosidase activity was monitored.

• At first, no -galactosidase activity because the Hfr donors lacked inducer and F- recipients were unable to to produce active enzyme.

• After 1 h conjugation when the I+Z+ enter the F- cells -galactosidase began and ceased after another hour.

• The donated Z+ gene entering the cytoplasm of the I- cell causes constitutive expression of the -galactosidase gene.

• After the I+ gene is expressed (after 1 h) it represses -galactosidase synthesis.

• I must be a diffusible product.

Figure 31-6 The PaJaMo experiment.

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F- strain resistant to T6 phage and streptomycin.

Figure 5-25 Control of transcription of the lac operon.

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Other constitutive mutations

• Second type of constitutive mutation in the lactose system is OC - operator constitutive.

• Independent of I gene-maps to space between I and Z genes.

• In a partially diploid F’ strain OC Z-/F O+ Z+, -galactosidase activity is inducible by IPTG.

• In the strain OC Z+/F O+ Z-, -galactosidase is constitutively produced.

Other constitutive mutations

• An O sequence can only control expression of the Z gene on the same chromosome.

1. The structural genes on DNA are transcribed onto complementary strands of mRNA.

2. The mRNAs transiently associate with ribosomes, which they direct in polypeptide synthesis.

The lac repressor binds to the O sequence to prevent the transcription of mRNA. In an OC mutant, the repressor cannot bind to the sequence.

Other constitutive mutations

• The simultaneous or coordinate expression of the lac enzymes under the control of a single operator indicates that the lac operon is transcribed as a single polycistronic mRNA

• Cistron-gene

• Control sequence that act only on the same DNA molecules as the genes they control are called cis-acting elements.

• Agents of diffusible products can be on different DNA molecules. Trans-acting factors.

RNA polymerase

• RNAP is the enzyme responsible for the DNA-directed synthesis of RNA.

• Enzyme couples together the ribonucleoside triphosphates ATP, CTP, GTP, and UTP on DNA templates in a reaction driven by the release and hydrolysis of pyrophosphate.

(RNA)n residues + NTP (RNA)n + 1 residues + PPi

• RNAP holoenzyme (459 kD) ’.

• Once RNA synthesis is initiated the subunit dissociates from the core enzyme ’which carries out the polymerization.

Table 31-1 Components of E. coli RNA Polymerase Holoenzyme.

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Figure 31-9 An electron micrograph of E. coli RNA polymerase (RNAP) holoenzyme attached to various

promoter sites on bacteriophage T7 DNA.

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RNA polymerase

• (+) strand (sense strand) or coding strand-has the same sequence as the transcribed RNA.

• (-) strand (antisense strand) acts as the template.

• By convention, the sequence template DNA is repesented by its template (nontemplate strand) so that it will have the same directionality as the transcribed RNA.

• RNAP holoenzyme binds to its initiation sites called promoters that are recognized by the corresponding factor.

• Promoters consist of ~40-bp sequence located 5’ to the transcription start site.

• Holoenzyme forms tight complexes with promoters (K ~10-14 M)

Promoter sequences

• In E. coli, most conserved sequence is a hexamer at about the -10 region (Pribnow box) of TATAAT.

• Upstream sequence at -35 is also highly conserved, TTGACA.

• Sequence unimportant between -35 and -10 but length is (16-19 bp).

• Initiating nucleotide (+1) is always A or G.

• Additional A+T rich sequence between -40 and -60 upstream promoter (UP) element binds to the C-terminal domain of RNAP’s subunits.

• Promoter mutations that increase or decrease the rate at which the associated gene is transcribed are called up mutations and down mutations.

Figure 31-10The sense (nontemplate) strand sequences of selected E. coli promoters.

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Figure 31-11a X-Ray structure of Taq RNAP core enzyme. subunits are yellow and green, subunit is

cyan, subunit is pink, subunit is gray.

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Figure 31-11b X-Ray structure of Taq RNAP. (b) The holoenzyme viewed as in Part a.

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Figure 31-12a The sequence of a fork-junction promoter DNA fragment. Numbers are relative to the

transcription start site, +1.

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Figure 31-12b X-Ray structure of Taq holoenzyme in complex with a fork-junction promoter DNA fragment.

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Figure 31-13a Model of the closed (RPc) complex of Taq RNAP with promoter-containing DNA extending

between positions –60 and +25.

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Figure 31-13b Model of the open (Rpo) complex of Taq RNAP with promoter-containing DNA showing the

transcription bubble and the active site.

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RNA polymerase

• RNAP holoenzyme (459 kD) ’.

• Crystal structure for Taq RNAP solved by Seth Darst.

• Active site has a Mg2+ ion.

• DNA template lies across one face of the enzyme outside the active site.

• Open and closed complexes.

• Closed complex has UP element contacts.

• Open complex, template strand of transcription bubble is in a tunnel formed by the ’subunits lined with basic amino acids.

• This tunnel leads to the active site.

Rifamycins inhibit prokaryotic RNAP

• Two related antibiotics: rifamycin B and rifampicin

• 2 X 10-8 M rifampicin inhibits 50% RNAP

• Binds to the subunit and prevents chain elongation.

Figure 31-14The two possible modes of RNA chain growth. Growth may occur (a) by the addition of

nucleotides to the 3 end and (b) by the addition of nucleotides to the 5 end.

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Chain elongation proceeds in the 5’ 3’ direction with RNAP• Experimentally proven with radiactively labelled [32-

P]GTP.

• For 5’ 3’ elongation, the 5’ -P is permanently labeled so that the chain’s level of radioactivity does not change upon replacement of labeled GTP with unlabeled GTP.

• For 3’ 5’ elongation, the 5’ -P would be added with each nucleotide, so that on replacement of labeled GTP by unlabeled GTP, the RNA chains lose their radioactivity.

• The 5’ 3’ elongation is observed experimentally, therefore, chain elongation proceeds 5’ 3’.

Figure 31-15RNA chain elongation by RNA polymerase.

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Figure 31-16An electron micrograph of three contiguous ribosomal genes from oocytes of the salamander Pleurodeles waltl undergoing

transcription.

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RNA polymerase cannot proofread

• Cannot rebind polynucleotide it has released.

• Enzyme is processive.

• No exonuclease activity.

• Error rate is one wrong base for every ~104 transcribed.

• DNA Pol I is one nt incorrect for every 107

• RNAP error rate is tolerable because most genes are repeatedly transcribed.

• The genetic code has synonyms (redundancy).

• Amino acid substitutions can be functionally innocuous.

• Large portions of many eukaryotic transcripts are excised when forming mature mRNAs.

Chain termination

• Transcriptional terminators share two common features:

1. A series of 4 - 10 consecutive A-Ts with the A’s on the template strand. The transcribed RNA is terminated in or just past this sequence.

2. A G-C rich region with a palindromic (2-fold) symmetric sequence that is immediately upstream of the series of A-Ts.

This sequence forms a self-complementary “hairpin” that is very stable.

Figure 31-18 A hypothetical strong (efficient) E. coli

terminator.

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Rho factor aids in termination

• Rho factor is a helicase that unwinds RNA-DNA and RNA-RNA double helices dependent on the hydrolysis of NTPs.

• Require a specific recognition sequence (80 -100 nt that lack a stable secondary structure and have multiple C rich regions, G poor regions) on the newly transcribed RNA upstream of the termination site.

• Attaches to nascent RNA at recognition site and migrates in the 5’ 3’ direction until it encounters RNAP paused at termination site and unwinds the RNA-DNA duplex that forms the transcription bubble.

• This releases the RNA transcript.

Figure 31-19a X-Ray structure of Rho factor in

complex with RNA. (a) The Rho protomer with its N-terminal domain cyan, its C-terminal

domain red, and their connecting linker yellow.

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Figure 31-19b X-Ray structure of Rho factor in complex with

RNA. (b) The Rho hexamer. Its six subunits, each of which are drawn in a different color, form an open lock washer-

shaped hexagonal ring.

Figure 31-19c X-Ray structure of Rho factor in complex with RNA. (c) The solvent-accessible surface of the Rho hexamer as viewed from the top of Part b.

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Control of transcription in prokaryotes

• Prokaryotes need to respond to sudden environmental changes such as the influx of nutrients, by inducing the synthesis of proteins.

• Transcription and translation are tightly coupled.

• Ribosomes commence translation near the 5’ end of the nascent mRNA soon after it is made by RNAP.

• Most prokaryotic transcripts are degraded within 1 - 3 min after their synthesis.

• In contrast, eukaryotic induction takes hours or days to respond because the transcription takes place in the nucleus and has to be exported to the cytoplasm for translation.

Figure 31-24An electron micrograph and its interpretive drawing showing the simultaneous transcription and

translation of an E. coli gene.

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Promoters

• The more the promoter resembles the consensus sequence, the stronger the promoter.

lac repressor binding

• lac repressor is a tetramer of 360 residue subunits which are each capable of binding one IPTG with a K = 10-6 M.

• In the absence of inducer, binds to duplex DNA nonspecifically (K = 10-4)

• Binds to the lac operator tightly (K = 10-13 M).

• Binds faster than diffusion rate constant in solution, so lac repressor slides along DNA quickly until it finds the lac operator sequence.

• lac operator sequence is nearly palindromic.

• lac repressor prevents RNAP from forming a productive initiation complex.

Figure 31-25 The base sequence of the lac operator.

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Figure 31-26The nucleotide sequence of the E. coli lac promoter–operator region.

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C-terminus LacI N-terminus LacZ

Catabolite repression

• Glucose is the carbon source of choice for E. coli, so if it is present in large amounts, the bacterium will suppress the expression of genes encoding proteins involved in other catabolites’ metabolism.

• This happens even when metabolites such as lactose, arabinose, or galactose are present in high concentrations.

• Catabolite repression-prevents the wasteful duplication of energy-producing enzymes.

Figure 31-27The kinetics of lac operon mRNA synthesis following its induction with IPTG, and of its

degradation after glucose addition.

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