Friday, November 1st Lecture Notes

Viruses are molecular parasites of cellular life

• Like cells, a virus has a nucleic genome that encodes its proteins

  • Different species of virus can have genomes that are made of RNA or DNA, and can be single-or double-stranded

• Unlike cells, a virus cannot reproduce in isolation

  • To propagate, the virus must infect a living cell and commandeer the host’s internal biochemistry

• Different organisms have distinct viral parasites

  • Viruses that infect bacteria are known as bacteriophages

For a bacteriophage, the initial steps of infection are binding of the viral particle to the host cell surface, and injection of the viral genome across the membrane into the cytoplasm

• The genome is the only part of the phage that actually enters the cell

Bacteria have evolved defense mechanisms that often destroy bacteriophages before they take over the cell

Bacteriophage Lambda

• Chromosome

  • 48 kb ds-DNA

  • 71 protein-coding genes

  • Linear chromosome has complementary 5’ overhangs at each end

    • Inside the bacterium, these sticky ends anneal and the chromosome becomes circular

• Since lambda’s discovery in 1950, lambda phage has been one of the most widely used systems for studying molecular biology

Bacteriophage Life Cycles

• This is a 2-way genetic switch; a phage must commit to either lysis or lysogeny

• Lytic cycle

  • Viral DNA is replicated repeatedly

  • Viral genes are used to synthesize large quantities of proteins

  • Viral DNA and proteins assemble into roughly 100 new viruses

  • Viral genes are released by disintegration of the cell

• Lysogenic cycle

  • Viral DNA becomes integrated into the bacterial chromosome

  • Viral DNA replicated with each subsequent cell division

Phases of Lysogeny

• Commitment

• Site-specific recombination of phage DNA into the bacterial chromosome

• Maintenance of the lysogenic prophage during bacterial growth and fission

• Exit from lysogeny and return to the lytic cycle

The decision between lysis and lysogeny is determined by transcriptional activity at 4 promoters in a 5 kb control region

Lysis and lysogeny result from two mutually exclusive patterns of gene expression

• Both patterns of expression occur during the commitment phase until one or other becomes dominant

Critical Genes in Lysis and Lysogeny

• The lambda control region contains two genes, cro and cl, that play a central role in the process of commitment

  • Expression of cro promotes lysis

  • Expression of cl promotes lysogeny

• The Cro transcription factor can bind to the already weak PRM promoter to repress the cl gene

• In addition to cro, transcription from the PR promoter also expresses genes that can carry out replication and lysis

• The cl gene can be transcribed from a second promoter called PRE

• The cll and cIII genes also contribute to the commitment, but both do so indirectly by regulating cl expression

• The CII protein is a transcription factor that can bind next to the weak PRE promoter

  • When CII is bound there, the PRE promoter is activated and the cI gene is transcribed

Strong Promoters

• PL and PR are strong constitutive promoters

  • The bacterial RNA Pol activates transcription at these promoters as soon as phage enters the cell

• Each of these promoters is used to transcribe a polycistronic operon

• The initial transcription from the strong PR promoter expresses both the cro gene and the cII gene

Weak Promoters

• PRM and PRE are weak promoters

  • They require activator proteins in addition to RNAP in order for a substantial level of transcription

    • Only these 2 weak promoters can be used to transcribe the cl gene

• The PRM and PR promoters overlap with three operator sequences (OR1-OR3) that can bind transcription factor proteins

  • A transcription factor or RNA Pol can be bound at the promoter at a given time, not both

• Cro protein has a high affinity for the OR3 operator

• When bound to OR3, Cro blocks the PRM promoter and represses cl transcription from that promoter

  • The PR promoter isn’t blocked and remains active

• Cl protein (lambda repressor) has its highest affinity for the OR1 operator

• When cl is bound to OR1, it blocks the PR promoter and represses cro transcription

When lambda first infects a bacterium, the strong viral promoters predominate and the cro gene gets a “head start” down the lytic pathway

The cro and cI genes encode transcription factors that can repress each other’s transcription

• Under some circumstances, cI has an advantage

Structure of cI protein

• Structure is critical to its function

• Each subunit contains:

  • An N-terminal region that has two distinct functional domains

    • A DNA-binding domain

    • An activation domain that can interact with RNAP

  • A C-terminal region that has distinct functional domains for dimerization and tetramerization of cI subunits

cI Protein

• Can bind to any of the three lambda operators, but it does so with differing affinities

  • cI has a high affinity for the ORI operator

  • cI has a 10-fold lower affinity for the OR2 and OR3 operators

• When cI protein is present at low concentrations, it is preferentially bound by the high affinity OR1 operator

• The cI protein is a HTH factor, and thus binds DNA as a homodimer

• Once a cI dimer is bond at the OR1 operator, a second cI dimer can bind efficiently at the OR2 operator because of cooperative binding between the transcription factors

  • They form a cI tetramer

• Once cI protein is bound at OR2, its activation domain can recruit RNA Pol to the weak PRM promoter and increase the rate of cI transcription

Cooperative binding occurs when:

• Two proteins can bind DNA independently

• When bound to DNA simultaneously, the two proteins also bind to one another

• This additional chemical bond increases the overall affinity and favors the bound state, thereby increasing the probability that both binding sites will be occupied

Important Concepts

• cI is an example of a transcription factor that functions as a repressor at one promoter (PR) but functions as an activator at a second promoter (PRM)

  • cI recruits RNA Pol to the promoter (acts as activator)

• When cI recruits RNA Pol to the PRM promoter, it is influencing its own synthesis, which is called autoregulation

If cI can establish autoregulation, it wins the competition and permanently represses cro

Positive autoregulation of cI

• Once the cI gene establishes this autoregulation, it represses both cro and cII

  • The cII protein is no longer needed once cI is being transcribed from the PRM promoter

If cI fails to establish autoregulation, then cro wins the competition and successfully represses cI

Mini Study Guide

• What are bacteriophage?

• Be able to explain the differences between the lytic and lysogenic life cycles.

• What are the four promoters that control life cycle choice and what genes do they control? Be able to draw the region

  • Which promoters are weak? Which ones are strong?

• How does Cro promoter the lytic pathway?

• How does cI promoter the lysogenic pathway?

  • For cro and cI: where do they bind and do they bind alone or in a complex?

• Is cI a repressor or an activator?

I-Clicker Questions

• Where does Cro bind, and what gene does it repress?

  • OR3, cI

• Which promoter does cII bind near, and which gene’s transcription does it activate?

  • PRE, cI

• Where does cI bind first, and which promoter does it obstruct?

  • OR1, PR

• When cI forms a tetramer, which binding sites are occupied?

  • OR1 and OR2

• When cI forms a tetramer and occupies both OR1 and OR2, what can occur?

  • cro expression is repressed

  • cI activates its own transcription through the PRM promoter

  • Activity of cII at the PRE promoter is no longer necessary

  • Autoregulation of cI

• A lambda phage undergoes a mutation such that the OR2 operator can no longer bind proteins. The other two operators and the core promoters are intact and functional. If this phage infects an E. coli, what effect will this mutation have on gene expression?

  • lack of cI autoregulation

  • inability to repress cro