Lecture 12- RNA Bacteriophages

Question 1

What are some reasons for studying RNA bacteriophages?

Answer 1

Extensively studied
Example of simplest virus
Provides basis for comparison with poliovirus
Model system for understanding regulation at level of translation
-genes either have translation turned on or off
-regulation: have discussed lac operon, which occurs at the level of transcription, but with RNA genome, no regulation occurs at the level of transcription

Question 2

Which viruses are members of the RNA Bacteriophage family?

Answer 2

MS2, f2, R17, and Qbeta

We will be looking at MS2

Question 3

The virion:

Answer 3

Capsid: icosahedral symmetry; T = 3.  180 identical structural subunits called the coat protein.  (capsid is the coat). 
RNA: single stranded, + polarity, 3569 nucleotides
A protein (393 aa); also called maturation protein and adsorption protein: one copy per virion; required for infectivity.  larger than coating protein.  A stands for adsorption.  Important for assembly process (required for assembly). Not covalently bonded, unlike terminal proteins!

Question 4

RNA phage particles pack into crystalline array

Answer 4

True

Question 5

Bacteriophage MS2 Structure

Answer 5

Triangulation number = 3

Has 5-fold and 6-fold axes of rotational symmetry

Question 6

Secondary Structure in MS2 RNA

Answer 6

There is a ton of secondary structure in this RNA; almost looks like tRNA or rRNA
The secondary structure contributes to compaction of RNA

Question 7

Non structural proteins in MS2

Answer 7

Replicase Protein (544 aa)

• RNA polymerase
• replicase requires the activities of three E coli proteins (Tu, Ts, and rib. protein S1- don’t memorize names though)
• replicase and e coli proteins form the active RNA pol?
• concept: most viruses hijack cellular proteins of the host. will use host components to generate functional proteins. all three proteins involved in translation in e coli

Lysis protein (75 aa)

Question 8

What are the structural proteins in MS2?

Answer 8

Capsid and A protein

Question 9

Adsorption in MS2

Answer 9

male-specific (b/c involves f-pilus); attaches to F- plus (binds to the side), requires A protein which is cleaved by a protease into 2 fragments
“Male” E coli carry a pilus, and this process requires that host carry a pilus; reason that adsorption is male specific. Genders refer to conjugation (DNA transferred from the male through the pilus to the female).
This binding requires the A protein. Believed that A protein is virion attachment protein in this process. A protein is cleaved by proteolysis into two fragments shortly after adsorption. We don’t know exactly how that adsorption occurs.
Others: bind to the tip of the pilus, not the side

Question 10

Entry in MS2

Answer 10

Virions probably move along pilus or pilus retracts until they encounter cell surface. Cleaved A protein accompanies RNA into cell (non covalently).
Key point: a single virus particle is sufficient for a complete infection. Therefore, incoming RNA genome must serve first as a template for translation and subsequently as a template for replication.

Question 11

Translation in MS2

Answer 11

Ribosomes attach to RNA genome at Shine-Dalgarno sequences to initiate translation. Once several ribosome are simultaneously translating an single RNA a polysome has been formed.
Polysome = RNA plus ribosomes that are translating it
Replication: cannot begin until after initial polysome is dismantled
Translation goes left to right, but replication goes right to left
Initially replicase binds to 3’ end. But how is the polysome dismantled?

Question 12

Genome Replication in MS2

Answer 12

Replicase binds to 3’ end of + RNA genome
Translates 5’-3’ (right to left)
- RNA = antigenome
Pol translates 5’-3’ but left to right, creating nascent + RNA chain

Question 13

Assembly in MS2

Answer 13

Psi= Packaging signal
Coat protein dimer binds at psi/packaging signal to initiate capsid process. Don’t understand how we go from this stage to full capsid with A protein associated with RNA. A protein required in some way because it’s the maturation protein.

Question 14

Lysis in MS2

Answer 14

Infected cells lyse after intracellular concentration of lysis protein exceeds some threshold level. Lysis occurs at about 60 minutes post-infection. Burst size is about 10,000 which is one of the largest we know of.
Lysis protein is made throughout infection, once it gets to a certain concentration the threshold bursts

Question 15

Genetic map of RNA phage MS2

Answer 15

Order: A protein, coat, replicase
Overlapping end of coat and beginning of replicase gene is the lysis gene
Lysis gene overlaps both the coat and replicase genes. Reading frame for lysis protein is DIFFERENT from coat and replicase reading frames. This is in the interest of economy. Coding region for lysis overlaps but it is still a single piece of RNA.
Untranslated region is important for replication process. Can use any of the many ribosome binding sites- this is important.

Question 16

Regulation of Gene Expression During RNA Bacteriophage Infection

Answer 16

1) Coat protein is synthesized throughout infection at a high rate; the rate increases with time
2) The A protein is synthesized after RNA synthesis begins and the rate increases with time. Approximately one A protein is made for each genome produced.
3) Lysis protein is synthesized at a low rate throughout infection.
4) Replicase is synthesized early, then shut off at late times (because you don’t need it at late times)
5) At an early time, the initial polysome is dismantled to allow RNA replication to begin
Test your understanding: end of studies, come back to this list and ask if you can account for each phenomenon listed.

Question 17

Mechanism Regulation of Translation

Answer 17

Two factors regulate the initiation of translation by limiting the accessibility of the ribosome binding site to the ribosome
1) secondary structure in the RNA
2) protein binding
All occurs at level of ribosome binding site. If rbs is accessible, will bind and translation will be initiated. If not, that gene is turned off b/c can’t be translated.

Question 18

Secondary Structural Elements in genome block some ribosome binding sites

Answer 18

Only accessible ribosome binding site with complete genome is the rbs for the coat gene. Note where termination codon for the coat gene is on diagram

Question 19

1) Coat Protein regulation

Answer 19

Coat protein synthesized throughout infection at a high rate; the rate increases with time.
Coat ribosome binding site is NOT blocked by secondary structure in the rna. Rate of synthesis increases as replication produces more + RNA templates.

Question 20

2) A protein Regulation

Answer 20

the A protein is synthesized after RNA synthesis begins and the rate increases with time. Approximately one A protein is made for each genome produced.
The A gene ribosome binding site is blocked by secondary structure in the complete + RNA genome. Synthesized after RNA synthesis begins. There is only one because rbs for A gene gets blocked. RBS will be accessible on nascent RNA chains during replication.
Ribosome gains entry to A gene ribosome binding site only when replicase has just finished synthesizing that region of the +RNA strand. Later as RNA synthesis proceeds, the secondary structure that blocks the ribosome binding site forms which then prevents any further translation of the gene. Gets longer, starts to fold, RBS blocked, no more translation of A protein. RBS accessible for JUST long enough. This mechanism results in approximately one A protein being synthesized for each + RNA made.

Question 21

2) Regulation of lysis protein

Answer 21

Synthesized at low rate throughout infection
The lysis gene ribosome binding site is inaccessible due to a secondary structure in the genome RNA. When the ribosome that is translating the coat gene terminates, it remains bound to the RNA and scans in both directions for an accessible rbs. Approximately 5% of the time it scans back and initiates at the lysis gene AUG.

Question 22

4) Replicase protein regulation

Answer 22

Synthesized early, then shut off at late times
Early in infection, replicase rbs accessible. Late in infection, not available. Coat protein dimer also binds to another site. Think about whether or not they might be the same site. Is the psi site the same as the rbs?

Question 23

Initial polysome dismantling

Answer 23

AT an early time, the initial polysome is dismantled to allow RNA replication to begin. Replicase protein binds to rbs for coat, no further ribosome binding. Ribosomes run off RNA. Double check this if confused.