Virus Entry

Chapter 8:
Bacteriophage Lambda

Again dealing with prokaryotic cells only

Bacteriophage Lambda

Virus Structure (complex)

- icosahedral (T=7)

(ds & linear)

- helical

Receptor
- J protein (binds to LamB)
- in E. coli outer memb.

Fig. 8.3 Schematic drawing of  virion.

1
 Genome Structure

- linear dsDNA, 40-60 kb Fig. 8.2 The genetic map of  DNA.

- complementary ssDNA ends (coshesive ends)

- encodes ~60 proteins (grouped by function)

- Transcriptional regulation of gene expression

(“Immediate Early”, “Early” & “Late” genes)

- transcription & genome replication by cellular Pol’s

(cDdRp) (cDdDp)

 Infection

Genome
circularizes
(cos ends)

prophage

Transcriptional Integration into

Regulation
bac. chromosome
Viral genome
(bac. = lysogen)
Replication
(active)
(uv)
Viral genome
Replication
(passive)

Fig. 8.1 Outline of  biology.

2
 Lytic OR Lysogenic

Metabolically- Metabolically-
Active Inactive
Cells Cells

Fig. 8.1 Outline of  biology.

Gene Expression (IMMEDIATE EARLY & EARLY)

cDdRp IMMEDIATE
EARLY (N, cro)

N Anti-term’s
PL and PR

EARLY
Fig. 8.4 The lytic transcription program. (Rec. & Rep.)

3
 Fig. 8.4 The lytic
Gene Expression (LATE) transcription program

EARLY (Q)

Q Anti-term’s
PR’

LATE
(Structural
& Lysis)

Lytic Program

PL & PR ON

IMMEDIATE
EARLY (N, cro)

EARLY
(Rec. , Rep.
& Q)

LATE
(Structural
& Lysis)

4
 What controls PL & PR ? Answer: CI

The CI Repressor Blocks the Lytic Program

PRM (Repressor Maintenance)

PRM is one of TWO promoters for CI

Fig. 8.4 The lytic
transcription program.

Prokaryotic Transcription

Promoter (P)

Initiation
Site (+1)
-35 -10

Operator A Pol Operator B

CI does both
Activation Repression

5
 PRM (Repressor Maintenance)

How CI Repressor Blocks the Lytic Program

OR1 > OR2  OR3 PR
Coop. binding
Bound: OR1 & OR2
Represses PR
v. v. weak  OFF
PR
Bound: OR1 & OR2 cro mRNA CI (dimer)
Activates PRM X
cI mRNA
strong
PL N mRNA
X Bound: OL1 & OL2
Represses PL

When OR3 is also (needs a

X cro mRNA
filled (at [CI]) long-range
cI mRNA Represses PRM interaction)
PR
Fig. 8.5 Transcription and its repression at PRM and PR. CI can A or R transcription
of it’s own mRNA from PRM
2

1
6
 Therefore CI shuts OFF Lytic Program by Repressing PL and PR

PL & PR Lytic Program

Immediate
Early (N, cro)

Early
(Rec. , Rep.
& Q)

Late
(Structural
& Lysis)

What controls the level of CI in infected cell? Answer: CII

CII is expressed from anti-terminated PR

(Early)

7
 CII functions as a Transcriptional Activator

CII activates a CII activates a 2nd

promoter for Int, PInt Promoter for CI , PRE

(Repressor
Establishment)

Recombinase inserts Antisense to mRNA-Q

prophage inducing Represses
lytic program CII activates a
lysogeny
(Activates PRM) Promoter for PAQ
Fig. 8.6 CII-activated promoters in lysogenization.

 Lysogenic

 [CII]

PL & PR  [CI]  [Int]

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  Lytic

 [CII]

PL & PR  [CI]  [Int]

Lytic OR Lysogenic

Metabolically Metabolically
 [CII] Active Inactive  [CII]
(less stable) Cells Cells (more stable)

9
 PRM not Activated
PL & PR not Repressed
cI not produced
(Lytic Program stays ON)
PRE not activated
PL & PR  IMMED. EARLY  Cro, N
LYTIC PROGRAM Metabolically
Active Cells  [cII]
Anti-term.
PL & PR  EARLY  Rec, Repl, Q, cII

Metabolically
Inactive Cells  [cII]
Anti-term.
PR’  LATE  Lysis, Structural
Activates
Inhibits Q
translation
( LATE genes)
PL & PR Repressed
(turns OFF Lytic Program)
PRE  cI Pint  Int PAQ  RNA-AQ
PRM is Activated (phage integrates)

Posting #4 LYSOGENIC PROGRAM

 Lysogenic   Lytic

CII induces
Pint  Int

Int
Int
Xis
(uv)

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  Lysogenic   Lytic
Bacterial Chromosome
uv
OFF

X
-35 -10
SOS

O ON
damaged
LexA RecA RecA

X
DNA
X

(repressor)

autoprotease

CI RecA [CI] =  PL & PR

X

(PL Xis, Int) = lytic growth

autoprotease

Excision

PL (early)

Fig. 8.2 The genetic map of  DNA.

1
11
 Integration & Excision

circular
Specific
sequences
attP – phage
attB – bacterial

uv = CI CII (meta. inactive) = CI

PL = ON Pint = ON PL = OFF
no Xis
Excision Integration
Proteins
Int, Xis – phage
IHF – bacterial
Fig. 8.7 Integration and excision of DNA. (bends DNA)

Genome Replication

PR (early)

Fig. 8.2 The genetic map of  DNA.

- O and P proteins regulate replication (not viral Pol’s)

- both are “early” genes expressed from anti-term. PR (by N)

- ori is the origin of replication

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 Genome Replication

- O protein binds ori and melts adjacent AT-rich region

- P protein recruits a bac. helicase called DnaB to ori

- bac. Pol’s do replicating: cDdDp, cDdRp (adds primers)

- Early Replication (before lytic/lysogenic decision): bi-directional

few cirular genomes
lytic mode
 linear copies of genome made by “Rolling Circle” replication

(e.g. HSV – Fig. 24.8)

(connected = concatemer)

cos cos cos cos cos cos

Fig. 8.2 The genetic map of  DNA.

Virus Assembly

PR’ (late)

- “late” genes expressed from anti-terminated PR’ (by Q)

many proteins involved

- e.g. some are incorporated intact
“ “ cleaved during incorporation
“ “ scaffolding (not incorporated)

(all considered STRUCTURAL proteins)

13
 concatemer Virus Assembly

- nicks opposite strands 12 bp apart

- virus head section is pre-assembled

- terminase/cos - bind to portal of prohead

(translocation by terminase)
- DNA packaging by energy-requiring process

- conformational change (leading to maturation)

- pre-assembled tail section binds to filled head

Fig. 8.9 DNA packaging and virion assembly.

Fig. 8.2 The genetic map of  DNA.

Cell Lysis

- “late” genes expressed from anti-terminated PR’ (Q)

- Three proteins involved: R - transglycosidase digest bac.

CW
Rz - endopeptidase

S - forms pore in CM so R and Rz

can get to peptidogylcan of CW

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 END of Chapter 8

Eukaryotic Viruses

Eukaryotic host Cells

15
 Eukaryotic DNA Replication

Fig. 5.21 (Alberts)

Eukaryotic RNA Transcription

RNA polymerase I: 5.8S, 18S, and 28S rRNA genes

RNA polymerase II: mRNAs, plus snoRNA and snRNA genes

RNA polymerase III: tRNA genes, 5S rRNA genes, some snRNA

genes and genes for other small RNAs

Table 6.2 (Alberts)

16
 Eukaryotic mRNA Transcription

General transcription factors

Fig. 6.16 (Alberts)

Eukaryotic mRNA Transcription

Specific
transcription factors

Fig. 6.19 (Alberts)

17
 Pre-mRNA splicing

Eukaryotic mRNA Structure

AUG UAG

5’UTR 3’UTR

Kozak sequence: (A/G)XXAUGG

-3 +1 +4
Fig. 6.22 (Alberts)
optimal

Eukaryotic Translation

eIF-4E / eIF-4G / PABP

complex

(i) circularizes mRNA

(ii) recriuts
40S ribo. subunit

40S scans 5’-to-3’

- looking for AUG
with “good”
Kozak sequence

Fig. 6.75 (Alberts)

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 NEXT:

Chapter 22
Papillomaviruses

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