FCH 532 Lecture 17

Extra Credit Assignment for Friday Mar. 2DeLisa seminar, 148 Baker, 3:00PMStudy guide 8 postedExam scheduled for Friday, March 9-will cover

up to translation (Chapter 32). Amino acid metabolism will be next exam

Chapter 31

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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cAMP is the signal molecule for lack of glucose

• cAMP is the signal molecule indicating a lack of glucose.

• In the presence of glucose, cAMP levels are diminished.

• Addition of cAMP overcomes catabolite repression by glucose.

• cAMP binding protein responsible for the action-catabolite activator protein (CAP); cAMP receptor protein (CRP).

• CAP is a homodimer of 210 residue subunits that undergoes large conformational change upon binding to cAMP.

• CAP-cAMP complex binds to the lac operon and stimulates transcription in the absence of lac repressor.

CAP-cAMP promotes high levels of expression for a weak promoter• CAP-cAMP complex binds to the lac operon and

stimulates transcription.

• CAP is a positive regulator-turns on transcription

• lac repressor is a negative regulator - turns off transcription

• lac operon has a weak (low-efficiency) promoter because it differs significantly from the consensus sequence.

• CAP interacts directly with RNAP via the C-terminal domain (CTD).

CTD binds to dsDNA nonspecifically but with higher affinity to A-T rich sites (UP elements).

Figure 31-28a X-Ray structures of

CAP–cAMP complexes. (a) CAP–cAMP in complex with a palindromic 30-bp

duplex DNA.

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Figure 31-28b X-Ray structures of CAP–cAMP complexes. (b) CAP–cAMP in complex with a 44-bp palindromic DNA and the CTD oriented similarly to

Part a.

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Figure 31-28c X-Ray structures of CAP-cAMP complexes. (c) CAP dimer’s two helix-turn-helix motifs

bind in successive major grooves of the DNA.

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CAP-dependent promoters

• Class I promoters (lac operon) require only CAP-cAMP for transcriptional activation. CAP binding site can be located at various distances on the DNA.

• Class II promoters also only require CAP-cAMP for transcriptional activation. CAP binding site only occupies a fixed position that overlaps the RNAP binding site.

• Class III promoters require multiple activators to maximally stimulate transcription. May be more than one CAP-cAMP complexes or a CAP-cAMP complex in concert with promoter specific activators.

DNA binding motifs

• CAP proteins form a supersecondary structure called a helix-turn-helix (HTH) motif that binds to DNA.

• HTF motifs associate with target base pairs mainly via side chains extending from the second helix of the HTH motif (recognition helix).

• HTH motifs are observed in the lac repressor, trp repressor, cI repressors, and Cro proteins from bacteriophages.

• Another type of structural motif observed in DNA binding proteins are -ribbons or two stranded anti-parallel b-sheets.

-ribbons are found in the met repressor (MetJ).

Figure 31-29 X-Ray structure of the N-terminal domain of 434 phage repressor-target DNA complex. (a) A skeletal model (b) HTH (2, 3) interaction with

target DNA (c) A space-filling model.

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Figure 31-30X-Ray structure of the 434 Cro protein in complex with DNA. (a) A skeletal model. (b) HTH (2,

3) interaction with target DNA (c) A space-filling model.

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Figure 31-31X-Ray structure of an E. coli trp repressor– operator complex.

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Figure 31-32a X-Ray structure of the E. coli met repressor- SAM-operator complex. (a) The overall

structure of the complex as viewed along its 2-fold axis of symmetry.

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Figure 31-32b X-Ray structure of the E. coli met repressor-SAM-operator complex. (b) The antiparallel

ribbon (yellow) in the DNA’s major groove.

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trp operon regulation.• Encodes 5 polypeptides that make 3 enzymes

mediating the synthesis of tryptophan from chorismate.

• Under control of trp repressor (homodimer, 107 residues)-binds L-tryptophan to form a complex that binds to the trp operator to reduce the rate of transcription.

• Trp forms a hydrogen bond to DNA phosphate group increasing the repressor-operator association (corepressor-acts in conjunction with trp repressor).

• Controls 2 other operons: trpR and aroH involved in chorismate synthesis.

trp operon attenuation• Transcriptional control through which bacteria

regulate the expression of certain operons involved in amino acid biosynthesis.

• Discovered with E. coli trp operon-before they thought it was just the trp repressor responsible for regulating operon.

• trp deletion mutants downstream of trpO increased trp operon expression 6-fold-additional transcriptional control elements.

• Sequence analysis revealed trpE is preceeded by a 162 nt leader sequence (trpL).

• The new control element is located in trpL ~30-60 nt upstream of trpE.

trp operon attenuation• When W is scarce, the entire 6720-nt

polycistronic trp mRNA, including trpL is synthesized.

• As W increases, rate of trp transcription decreases as a result of the trp-repressor-corepressor complex.

• Of the trp mRNA that is transcribed, an increasing amount consists of only a 140-nt segment corresponding to the 5’ end of trpL.

• The availability of tryptophan results in the premature temination of the trp operon transcription.

• The control element responsible is an attenuator.

Figure 31-39A genetic map of the E. coli trp operon indicating the enzymes it specifies and the reactions

they catalyze.

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Figure 31-40The base sequence of the trp operator. The nearly palindromic sequence is boxed and its –10

region is overscored.

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trp operon attenuation: mechanism• The attenuator transcript has 4 complementary

segments that form one of two sets of mutually exclusive base paired hairpins.

• Segments 3 and 4 together with the succeding residues make a normal rho-independent transcription terminator: G-C rich sequence that forms a hairpin with several sequential U residues.

• Transcription rarely proceeds beyond this termination site when W is scarce.

• A section of the leader sequence (segment 1) is translated to form a 14-residue polypeptide with 2 consecutive Trp residues.

• This provides a clue to the mechanism.

trp operon attenuation: mechanism• When an RNAP that has escaped repression

initiates the trp operon transcription, a ribosome attaches the ribosomal initiation site of trpL mRNA and begins translation of the leader peptide.

• When W is abundant, lots of tryptophanyl-tRNATrp, the ribosome follows closely behind the transcribing RNA polymerase to sterically block the formation of the 2-3 hairpin.

• The prevention of the 2-3 hairpin allows the formation of the 3-4 hairpin which results in the termination of transcription.

• If there are low levels of Trp, the ribosome stalls on the 1 position and the 2-3 antiterminator forms allowing transcription of the trp operon.

Figure 31-41The alternative secondary structures of trpL mRNA.

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Figure 31-42a Attenuation in the trp operon. (a) When tryptophanyl–tRNATrp is abundant, the ribosome

translates trpL mRNA.

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Figure 31-42b Attenuation in the trp operon. (b) When tryptophanyl–tRNATrp is scarce, the ribosome

stalls on the tandem Trp codons of segment 1.

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Table 31-3 Amino Acid Sequences of Some Leader Peptides in Operons Subject to Attentuation.

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Regulation of rRNA synthesis: Stringent Response• Under optimal conditions, E. coli divides every 20

min.

• These cells contain up to 70,000 ribosomes so 35,000 ribosomes must be made per cell division.

• RNAP can initiate transcription at 1 gene per sec.

• In order to meet the needs of the cell for ribosomes, there are multiple copies (7) or the rRNA operon in the E. coli genome.

• Rapidly growing cells contain multiple copies of their replicating chromosomes.

• The rate of rRNA synthesis is proportional to the rate of protein synthesis.

• Stringent response: a shortage of any species of amino acid charged tRNA that limits the rate of protein synthesis triggers a metabolic adjustment.

Stringent Response• Stringent response: a shortage of any species of

amino acid charged tRNA that limits the rate of protein synthesis triggers a metabolic adjustment.

• Can cause a 10 to 20-fold reduction in the rate of rRNA and tRNA synthesis.

• Stringent control depresses numerous metabolic processes (DNA replication, biosynthesis of carbohydrates, lipids, nucleotides, proteoglycans, glycolytic intermediates) while stimulating other pathways (amino acid biosynthesis).

Stringent Response• 2 nucleotides regulate the stringent response

ppGpp and pppGpp. Together known as (p)ppGpp.

• The accumulation and decay of these regulates the stringent response.

• Relaxed control mutants designated relA-, do not exhibit the stringent response-lack (p)ppGpp.

• (p)ppGpp inhibits the transcription of rRNA genes but stimulates transcription of the trp and lac operons.

• Stringent factor (RelA) catalyzes the reaction:

ATP + GTP AMP + pppGpp

ATP + GDP AMP + ppGpp

Stringent Response• Several ribosomal proteins convert pppGpp to ppGpp.

• Stringent factor is only active in association with a ribosome that is actively engaged in translation.

• (p)ppGpp synthesis occurs when ribosome binds its mRNA specified but uncharged tRNA.

• The binding of a specified and charged tRNA greatly reduces the rate of (p)ppGpp synthesis.

• (p)ppGpp degradation is catalyzed by the spoT gene product.

Eukaryotic RNA polymerases

1. RNA polymerase I (RNAP I, Pol I, RNAP A)-located in nucleoli, synthesizes rRNA precursors.

2. RNA polymerase II (RNAP II, Pol II, RNAP B)- in the nucleoplasm, synthesizes mRNA precursors.

3. RNA polymerase III (RNAP III, Pol III, RNAP C)- alos in nucleoplasm, synthesizes precursors of 5S rRNA, tRNAs, and other small nuclear and cytosolic RNAs.

Have greater subunit complexity than prokaryotic RNAP.

Molecular masses up to 600 kD

Table 31-2 RNA Polymerase Subunitsa.

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RNAP II• RNAP II is regulated by it’s C-terminal domain (CTD).

• Contains 52 highly conserved repeats of the heptad PTSPSYS in mammals (26 in yeasts).

• Subject to phosphyorylation/dephosphorylation by CTD kinases and CTD phosphatases.

• RNAP II initiates transcription when CTD is unphosphorylated.

• RNAP II commences elongation only after the CTD is phosphorylated.

• Crystal structures shows it is similar to the Taq RNA polymerase.

• RNAP II binds 2 Mg2+ ions in the active site.

• Several subunits not observed in Taq RNA polymerase.

Figure 31-20a The X-Ray structure of yeast RNAP II that lacks its Rpb4 and Rpb7 subunits.

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Figure 31-20b The X-Ray structure of yeast RNAP II that lacks its Rpb4 and Rpb7 subunits. (b) View of the enzyme from the right in Part a showing its DNA binding

cleft.

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RNAP II• Eukaryotic RNAPs cannot independently bind to their

target DNA.

• They must be recruited to target promoters through complexes of transcription factors.

• RNAP II can initiate transcription on a dsDNA with a 3’ single-stranded tail at one end.

Figure 31-21a Secondary structure of an RNAP II elongation complex. Template DNA cyan, nontemplate

DNA green, and newly synthesized RNA red.

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Figure 31-21c Cutaway schematic diagram of the transcribing RNAP II elongation complex.

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Amatoxins• Amanita phalloides (death cap) mushroom produces

bicyclic octapeptides known as amatoxins. -amanitin is shown:

• Forms a tight 1:1 complex with RNAP II (K = 10-8 M) and RNAP III (K = 10-6 M).

• Binding slows RNAP synthesis from 1000s to a few nt per min.

• RNAP I, mitochondrial, chloroplast and prokaryotic RNAPs are insensitive.

Figure 31-22The proposed transcription cycle and translocation mechanism of RNAP. (a) Nucleotide addition cycle. (b) RNA · DNA complex in RNAP II.

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Eukaryotic promoters• Mammalian RNA polymerase I has a bipartite promoter

consisting of a core promoter element (-31 to +6) and upstream promoter element (-187 to -107) ; GC-rich, recruits transcription factors.

• RNA polymerase II promoters are longer than prokaryotic promoeters.

• Constitutively expressed genes have 1 or more copies of GGGCGG (GC box) located upstream of transcription start site.

Eukaryotic promoters• Most structural genes have a conserved AT-rich

sequence 25-30 bp upstream from transcription start site.

• TATA box (sometimes called Hogness box)-resembles -10 region of prokaryotic promoters.

• Deletion of TATA box does not eliminate transcription; instead generates differences in transcription start site.

• -50 to -110 also contains promoter elements, example: globin genes have a conserved CCAAT box -70 to -90.

• Globins also have the CACCC box upstream from the CCAAT box.

Figure 31-23 The promoter sequences of selected

eukaryotic structural genes.

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Enhancers in eukaryotes• Enhancers are transcriptional control regions that can

be located several thousand base pairs upstream or downstream from the transcription start site.

• Enhancers must be associated with promoters to trigger site-specific and strand -specific transcription initiation.

• Required for full activities from promoters.

• Enhancers are recognized by specific transcription factors that stimulate RNA polymerase II to bind to the corresponding but distant promoters.

• Mediate selective gene expression in eukaryotes.

RNA processing

• Most (all in eukaryotes) primary transcripts (the RNA molecule as encoded in the DNA) function after being altered covalently by one or more of the following processing steps: removal of 5’ and/or 3’ nucleotides, addition of nucleotides at the 5’ and/or 3’ ends, covalent modification of the bases, or “editing” of the nucleotide sequence (changing the information content of the RNA).

• Require one or more of the following activities: capping enzyme, polyA polymerase, specific ribonuclease, RNA ligase, spliceosomes-specialized RNA processing complexes consisting RNA and protein, and catalytic RNA-ribozymes

Messenger RNA processing in eukaryotes

• 1. Capping

• 2. Polyadenylation

• 3. Splicing

• 4. Introns early or introns late?

A gene is not necessarily co-linear with its encoded protein (for eukaryotic genes only)

The linear order is never violated; it is simply interrupted

AUG … UAG 3’ UTR

5’ UTR

Green=ORF(open reading frame)

• 5’ cap is a reversed guanosine residue so there is a 5’-5’ linkage between the cap and the first sugar in the mRNA.

• Guanosine cap is methylated. (cap-0)

• First (cap-1)and second nucleosides (cap-2) in mRNA may be methylated

Eukaryotic mRNA is capped

Capping mRNA-cont.

• Capping involves several enzymatic reactions

1. Removal of the leading phsophate group from the 5’ terminal triphosphate group by RNA triphosphatase

2. Guanylation of the mRNA by capping enzyme; requires GTP and yields the 5’-5’ triphosphate bridge and PPi

3. Methylation of guanine by guanine-7-methyltransferase (methyl from SAM).

4. The O2’ methylation of mRNAs first and maybe second nucleotide by a SAM requiring 2’-O-methyltransferase.

Capping enzyme and guanine-7-methyltransferase bind to phosphorylated CTD of RNAPII.

Poly (A) tails

• Eukaryotic mRNAs are monocistronic.• Sequences signaling transcriptional termination not

identified; not precise.• Mature mRNAs have well defined 3’ ends of poly A tails

(~250 in mammals and ~80 in yeast).• Added in two reactions by a complex of at least 6 proteins.

Polyadenylation of eukaryotic mRNA• Transcript is cleaved to yield a free 3’-OH group at a

specific site 15-25 nt past an AAUAAA site and within 50 nt before U-rich or G-U rich sequence

• Endonuclease that cleaves RNA uncertain but requires cleavage factors (CFI and CFII).

• Poly(A) tail is made from ATP by poly(A) polymerase (PAP) which is recruited by the cleavage and polyadneylation specificity factor (CPSF).

Polyadenylation -cont.• Polyadenylation is correlated with messenger

half-life:• No/short polyA>short mRNA life-time; long

polyA>long life-time of mRNA.• Short half-life:little protein; long half-life, more

protein

Purification of messenger RNA using oligo dT columns

•Why does polyA anneal to oligo(dT) at room temp?

Oligo(dT) attached to cellulose

Total cellular RNA;apply at room temperature to anneal polyA tail to oligo(dT)

} non-polyARNA flowsthrough

AAAA..TTTT

5’

polyA binds to oligo(dT) oncolumn

65o

C

TTTT

AAAAA..

polyA mRNA elutes at high temperature

Messenger RNA splicing• Pulse-chase labeling studies indicated

two important characteristics of eukaryotic RNA:

• 1. Most of the rapidly-synthesized RNA in the nucleus never reached the cytoplasm.

• 2. The rapidly-synthesized nuclear RNA was much larger on average than cytoplasmic RNA.

Cells + 32P phosphate for30 seconds (“pulse”)

32P-RNA

+31P for 10 minutes (“chase”)

32P-RNA

A pulse-chase experiment

Heteroduplex analysis-the first evidence of splicing

• The annealing of viral DNA also occurred

Viral DNA

denatureVirus-infected cells

Isolate mRNA

Anneal, shadow with heavy metals,analyze in EM

Heteroduplex analysis-continued

• The appearance of D-loops (displacement loops) in the DNA-RNA hybrid indicated the presence of regions of DNA that were transcribed, but later discarded from the RNA product.

Expected:

Observed:

Intron sequences not present inmature mRNA

Eukaryotic genes: alternating expressed and unexpressed sequences

• Most eukaryotic genes are intersperesed with unexpressed regions.• Primary sequences vary greatly in length (~2000 - 20,000 nt); much

larger than expected based on the proteins encoded-heterogeneous nuclear RNA (hnRNA).

• premRNAs are processed by the excision of internal sequences (introns) which can be 4-10 times longer in aggregate length than the expressed seqeuences (exons).

DNA-RNA heteroduplexes

• Annealing RNA from virus-infected cells with viral DNA revealed the existence of seven introns-transcribed regions of the DNA removed from the mature mRNA. How would you prove the loops were DNA and not RNA?

Interpretation of the EM image: