Editing Lambda phage (section)

=== Infection ===
[[File:MANXYZ permease Step 4.jpg|thumb|450px|right|upright|Lambda phage J protein interaction with the LamB porin]]

Lambda phage is a non-contractile tailed phage, meaning during an infection event it cannot 'force' its DNA through a bacterial cell membrane. It must instead use an existing pathway to invade the host cell, having evolved the tip of its tail to interact with a specific pore to allow entry of its DNA to the hosts.
# Bacteriophage Lambda binds to an ''E. coli'' cell by means of its J protein in the tail tip. The J protein interacts with the maltose outer membrane [[porin (protein)|porin]] (the product of the ''lamB'' gene) of ''E. coli'',<ref>{{cite journal | vauthors = Werts C, Michel V, Hofnung M, Charbit A | title = Adsorption of bacteriophage lambda on the LamB protein of Escherichia coli K-12: point mutations in gene J of lambda responsible for extended host range | journal = Journal of Bacteriology | volume = 176 | issue = 4 | pages = 941–947 | date = February 1994 | pmid = 8106335 | pmc = 205142 | doi = 10.1128/jb.176.4.941-947.1994 }}</ref> a porin molecule, which is part of the [[maltose]] operon.
# The linear phage genome is injected through the outer membrane.
# The DNA passes through the mannose permease complex in the inner membrane<ref>{{cite journal | vauthors = Erni B, Zanolari B, Kocher HP | title = The mannose permease of Escherichia coli consists of three different proteins. Amino acid sequence and function in sugar transport, sugar phosphorylation, and penetration of phage lambda DNA | journal = The Journal of Biological Chemistry | volume = 262 | issue = 11 | pages = 5238–5247 | date = April 1987 | pmid = 2951378 | doi = 10.1016/S0021-9258(18)61180-9 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Liu X, Zeng J, Huang K, Wang J | title = Structure of the mannose transporter of the bacterial phosphotransferase system | journal = Cell Research | volume = 29 | issue = 8 | pages = 680–682 | date = August 2019 | pmid = 31209249 | pmc = 6796895 | doi = 10.1038/s41422-019-0194-z }}</ref>  (encoded by the manXYZ genes) and immediately circularises using the ''cos'' sites, 12-base G-C-rich cohesive "sticky ends". The single-strand viral DNA ends are ligated by host [[DNA ligase]].  It is not generally appreciated that the 12 bp lambda cohesive ends were the subject of the first direct nucleotide sequencing of a biological DNA.<ref name="src3"/>

[[File:MANXYZ permease Step 10.jpg|thumb|350px|right|Lambda phage DNA injection into the cell membrane using Mannose PTS permease (a sugar transporting system) as a mechanism of entry into the cytoplasm]]

# Host [[DNA gyrase]] puts negative [[supercoil]]s in the circular chromosome, causing A-T-rich regions to unwind and drive transcription.
# Transcription starts from the constitutive ''P<sub>L</sub>'', ''P<sub>R</sub>'' and ''P<sub>R'</sub>'' [[promoter (biology)|promoters]] producing the 'immediate early' transcripts. At first, these express the ''N'' and ''cro'' genes, producing N, Cro and a short inactive protein.

[[File:N protien.svg|thumb|200px|Early activation events involving N protein]]

# Cro binds to ''OR3'', preventing access to the ''P<sub>RM</sub>'' promoter, preventing expression of the ''cI'' gene. N binds to the two ''Nut'' (N utilisation) sites, one in the ''N'' gene in the ''P<sub>L</sub>'' reading frame, and one in the ''cro'' gene in the ''P<sub>R</sub>'' reading frame.
# The N protein is an [[antiterminator]], and functions by engaging the transcribing [[RNA polymerase]] at specific sites of the nascently transcribed mRNA. When [[RNA polymerase]] transcribes these regions, it recruits N and forms a complex with several host Nus proteins. This complex skips through most termination sequences. The extended transcripts (the 'late early' transcripts) include the ''N'' and ''cro'' genes along with ''cII'' and ''cIII'' genes, and ''xis'', ''int'', ''O'', ''P'' and ''Q'' genes discussed later.
# The cIII protein acts to protect the cII protein from proteolysis by FtsH (a membrane-bound essential ''E''. ''coli'' protease) by acting as a competitive inhibitor. This inhibition can induce a [[bacteriostatic]] state, which favours lysogeny. cIII also directly stabilises the cII protein.<ref>{{cite journal | vauthors = Kobiler O, Rokney A, Oppenheim AB | title = Phage lambda CIII: a protease inhibitor regulating the lysis-lysogeny decision | journal = PLOS ONE | volume = 2 | issue = 4 | pages = e363 | date = April 2007 | pmid = 17426811 | pmc = 1838920 | doi = 10.1371/journal.pone.0000363 | doi-access = free | bibcode = 2007PLoSO...2..363K }}</ref>
On initial infection, the stability of [[CII protein|cII]] determines the lifestyle of the phage; stable cII will lead to the lysogenic pathway, whereas if [[CII protein|cII]] is degraded the phage will go into the lytic pathway. Low temperature, starvation of the cells and high [[multiplicity of infection]] (MOI) are known to favor lysogeny (see later discussion).<ref>{{cite book |last1=Henkin |first1=Tina M. |last2=Peters |first2=Joseph E. |title=Snyder and Champness molecular genetics of bacteria |date=2020 |publisher=John Wiley & Sons, Inc |location=Hoboken, NJ |isbn=9781555819750 |pages=293–294 |edition=Fifth |chapter=Bacteriophages and Transduction}}</ref>

==== N antitermination ====
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|footer =N Antitermination requires the assembly of a large ribonucleoprotein complex to effectively prolong the anti-termination process, without the full  complex the RNA polymerase is able to bypass only a single terminator<ref name="Korlach 2008">{{cite journal | vauthors = Santangelo TJ, Artsimovitch I | title = Termination and antitermination: RNA polymerase runs a stop sign | journal = Nature Reviews. Microbiology | volume = 9 | issue = 5 | pages = 319–329 | date = May 2011 | pmid = 21478900 | pmc = 3125153 | doi = 10.1038/nrmicro2560 }}</ref>

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This occurs without the N protein interacting with the DNA; the protein instead binds to the freshly transcribed mRNA. Nut sites contain 3 conserved "boxes", of which only BoxB is essential.
# The boxB RNA sequences are located close to the 5' end of the pL and pR transcripts. When transcribed, each sequence forms a hairpin loop structure that the N protein can bind to.
# N protein binds to boxB in each transcript, and contacts the transcribing RNA polymerase via RNA looping. The N-RNAP complex is stabilized by subsequent binding of several host Nus (N utilisation substance) proteins (which include transcription termination/antitermination factors and, bizarrely, a ribosome subunit).
# The entire complex (including the bound ''Nut'' site on the mRNA) continues transcription, and can skip through termination sequences.