7/3/01

Lecture #5 - Bacteriophage l

l basic facts

• l is a model organism for molecular biology. It has been studied from the late 1940s to the present.
• l is a bacteriophage - a virus that infects bacteria (in this case, E. coli)
• has a proteinaceous head and tail
• head contains ds linear DNA
• genome is 48 kb in length- encodes 40 genes
• 21 structural genes (head and tail)
• 19 genes for regulation of infection cycle
• l dependent on host enzymes for growth (uses host RNAP and DNAP). So, l promoters have the usual &endash;10 and &endash;35 conserved regions. This is in contrast to bacteriophage T7, which encodes its own RNAP that recognizes different promoter sequences.

l is a temperate phage- has two life cycles

1. lytic cycle- the classic mechanism of viral infection

• infection, production of 100-200 new viral particles, lyse host cell and infect new cells
• exponential growth of infectious particles

2. lysogenic cycle- l integrates its DNA into E. coli chromosome

• integrated l DNA called a prophage
• l chromosome replicates with E. coli chromosome
• prophage can remain in this state, or can be induced ( l DNA is excised) in response to certain signals and return to lytic cycle

Figure 11.1- l life cycle

• l lands on cell surface, injects its DNA
• lytic- l takes over cell machinery, goes on to infect more cells, orŠ
• lysogeny as a prophage. Need induction to return to lytic cycle. l lysogens are immune to further infection by l.

l Growth

• grow a lawn of bacteria in a petri dish
• small circular regions where bacteria not growing- phage have infected and lysed cells
• absence of bacterial growth = plaque = l growth
• wt l causes turbid plaques (mixed lytic and lysogenic growth)
• mutant l can undergo strictly lytic growth. Get clear plaque phenotype (no growth in middle of plaque). Mutations in genes cI, cII, cIII --> clear plaques.

l regulation- complicated! Why study l?

1. Model for temporal control of gene expression

• early genes --> late genes (regulated cascade of gene expression / regulation)

2. Model developmental system

• l - lytic or lysogenic
• insights into how eukaryotic cells differentiate during development

Figure 11.1- temporal control of gene expression

• early- certain genes transcribed early, including enzymes for DNA synthesis and DNA replication.
• late- structural genes transcribed
• packaging and lysis- l particles self-assemble (DNA, head, and tail form an infectious particle)
• timing is critical - don't want new virus made until ready to lyse cell

Immediate early genes

• N - transcribed from pL promoter
• cro - transcribed from pR promoter
• no genes initially transcribed from promoter pR', though pR' is a strong promoter

cro- negative regulator of lysogenic cycle

• (-) regulator of cI (encodes l repressor)
• favors lytic growth

N - antiterminator protein

• regulated readthrough of transcription terminators
• specific for certain transcription units
• effect of N- readthrough of tR1, tR2, tL1, tL2
• antitermination functions via sequences called nut sites (N-utilization sites)
• 2 such sites (nutL and nutR) are in DNA and encoded into RNA. Probably the RNA sequence is the recognition element.

In the presence of N, transcription takes place through nut site to make RNA. N gains access to and interacts with transcription machinery here - termination of transcription no longer takes place, so additional genes transcribed (cII and Q from pR; cIII from pL). This leads to production of delayed early genes.

Delayed early genes

• 2 needed for DNA replication
• 7 recombination genes (lysogeny is a site-specific recombination event) - int and xis genes
• 3 regulators

  1. cII- positive transcriptional activator

  • unstable protein- short 1/2 life- rapidly degraded
  • activates initial expression of cI ( l repressor)

  2. cIII- stabilizes cII at protein level

  • protects cII protein from degradation --> longer 1/2 life

  3. Q- antiterminator

  • turns on late genes - causes regulated read through of tR'
  • functions via specific sites called qut sites (Q-utilization sites)
  • favors lytic growth - Q gains access to transcription machinery, read through occurs therefore genes for lysis, head, and tail transcribed

Other regulators

1. cro- immediate early gene

• favors lytic cycle

2. cI- l repressor

• necessary for establishment and maintenance of lysogenic growth
• only gene required for maintenance of lysogenic growth

cI - l repressor- three regulatory activities

1. (-) regulator of transcription from pR and pL

2. (+) regulator of pRM ("repressor maintenance") - cI controls its own expression through a positive autoregulatory loop

3. (-) regulator of pRM - negative autoregulatory loop

cro - lytic growth

1. (-) regulator of pRM

2. (-) regulator of pR, pL - only when high [cro]

cII - (+) regulator leading to higher levels of transcription of genes important for lysogenic growth

1. pRE - "repressor establishment"

2. pINT - int codes for integrase (site-specific recombination of l into E. coli chromosome)

3. pANTI-Q - antisense of Q gene

Lysogenic cycle

• cII and cIII positively regulate cI (which has (+) or (-) autoregulation)
• cII and cIII necessary for establishment of lysogeny
• cI necessary for establishment and maintenance of lysogeny

Lytic cycle

• Q and cro lead to expression of late genes - head, tail, lysis
• if late genes transcribed, have lytic cycle - self-assembly of viral particles

If all the immediate and delayed early proteins are present in E. coli, which cycle will occur? It is a fine balance.

l control region - immunity region

OR and OL - operators with similar but not identical sequences

• OL separated into suboperators OL1, OL2, and OL3
• OR separated into suboperators OR1, OR2, and OR3

What proteins bind to these operators?

• cro and cI
• proteins are similar in 1°, 2°, and 3° structure, but are not identical
• have different DNA binding affinities
  • cro: OR3 > OR2 = OR1, and OL3 > OL2 = OL1
  • cI: OR1 > OR2 = OR2, and OL1 > OL2 = OL3
• cro and cI bind to DNA as dimers
• dyad symmetry in operators
• each monomer recognizes a 1/2 site of the suboperators

Lysogenic growth

• pRE is a weak promoter, i.e. has poor match to consensus sequences at &endash;10 and &endash;35 and thus requires (+) activator- cII protein (delayed early gene)
• remember that cIII proteins stabilizes the cII protein
• from pRE, antisense cro is transcribed - can't be transcribed into cro protein. From pR, cro is transcribed, therefore have two complementary RNA stands base pair, inhibiting translation of cro. This is down regulation of the lytic cycle.
• from pANTI-Q, antisense Q, RNA is transcribed. Get down regulation as above. Also called "antisense" regulation.

cI can act as a (+) or (-) regulator of pRM

• with low to intermediate [cI], then cI acts as (+) regulator
• with high [cI], cI acts as (-) regulator
• these are autoregulatory loops

Figure 11.25 - cII protein

• (+) regulator of pRE promoter
• weak promoter- non-consensus sequences at &endash;10 and &endash;35 (only 3 of 6 bases match consensus at each position)
• footprinting experiment - cII binds upstream of RNAP, and overlaps a little. cII probably helps RNAP recognize promoter. cII stimulates closed complex and open complex formation with 100-fold effect.

Figure 11.22 - complex operators OL and OR

note: in figure, pL / OL is flipped to be in same orientation as pR / OR

1. each operator has dyad symmetry

• OR1 and OL1 are more similar than OR1 and OR3 - makes sense because recognized by l repressor
• OL3 similar to OR3

2. proteins bind to OR1 and OL1 - overlaps the &endash;10 sequence and block

3. binding l repressor to OR2 stimulates transcription of pRM

4. binding to OR3 blocks transcription of pRM

5. no ribosome-binding site for transcript made from pRM; the first three bases of the RNA are AUG (initiating Met). Usually a ribosome binding site (Shine-Delgarno sequence) 5' to the AUG of initiating Met - not present here, so this RNA not translated efficiently. By contrast, the transcript initiated at pRE has a good ribosome binding site for cI and is translated efficiently.

Cooperative binding

• occurs at OR1 and OR2, and OL1 and OL2
• once one molecule of l repressor is bound to OR1, very likely that second molecule of cI will bind to OR2. The end result it that there is almost simultaneous binding of cI to OR1 and OR2
• binding to OR3 not cooperative
• cro does not exhibit cooperative binding, only cI does

l repressor

• cI binds to OR1 and OR2 - no transcription from pR (blocking &endash;10 and &endash;35 regions). But binding to OR2 stimulates transcription from pRM - (+) regulation
• in l lysogen, only cI gene expressed

Lytic cycle

• Q antiterminator - a delayed early gene. Transcription initiates at pR' (a strong promoter), but get readthrough of tR' because Q functions at qut site. Transcription of lysis, head, and tail genes takes place.
• cro initiated from pR - cro a (-) regulator of pRM ("repressor maintenance")
• cro therefore represses lysogeny

Hard to tell which cycle will win out.

• cII protein- unstable, short 1/2 life
• cIII - stabilizes cII
• proteins that degrade cII are encoded by E. coli
• key gene is Hfl gene which encodes Hfl protease
  • lots of Hfl protease --> little cII (lytic)
  • little Hfl protease --> lots of cII (lysogenic)
  • l monitors the level of Hfl
• Production of Hfl protease dependent on E. coli growth conditions
  • 1. rich nutrient media results in highly active Hfl protease favoring lytic growth
  • 2. poor nutrient media results in less Hfl protease, resulting in more cII favoring lysogenic growth

To induce lytic cycle from a lysogen, need to get rid of l repressor. Need more than starvation of host - DNA damage needed.

Immunity

l lysogens "immune" from infection by wt l

cI (made from lysogen) binds to OR1 and OL1 of incoming wt l - blocks transcription of pR and pL