Lecture 10

Question 1

Regulating translation initiation:

Answer 1

• Protein binds mRNA regulating initiation at SD/AUG (influencing ribosomal binding)
• sRNA binds mRNA regulating initiation at SD/AUG
• Structure in mRNA inhibits initiation at SD/AUG, could be a small MW effector molecule or a ribosome translating an upstream ORF.

Question 2

Translation:

Answer 2

• Most ORF have their own SD sequence with the initiation codon at the start
• Each ORF is translated by independent ribosomes

Question 3

SD sequence:

Answer 3

• Complementary to the 3’ end of the 16s RNA in the ribosome, so it positions the ribosome ready for translation

Question 4

General mechanisms for regulation translation initiation:

Answer 4

• AN RNA-binding protein prevents translation initiation by blocking the ribosome site
• A secondary structure in the mRNA prevents translation initiation of the second ORF, so translation of the first ORF disrupts the structure allowing translation.

Question 5

Ribosomal proteins:

Answer 5

• Large subunit
• Small subunit
• Expressed in a series of different operons

Question 6

Regulation of ribosomal proteins:

Answer 6

• Some ribosomal proteins interact with rRNA forming an integral structure.
• If rRNA is in excess, proteins will be used.
• If ribosomal proteins are in excess, protein 2 binds to mRNA preventing translation of protein 1.
• Translation of ORF2 is inhibited by structure in mRNA

Question 7

eg) Ribosomal S8 normally binds structure A in the 16S rRNA:

Answer 7

• If in excess, ribosomal protein S8 binds structure B in the mRNA encoding S9 thus inhibiting translation of itself
• Feedback regulation

Question 8

Riboswitches:

Answer 8

• can regulate translation initiaition and influence transcription termination
  eg) SAM is a small MW effector molecule that alters the structure of mRNA to block the ribosome binding site, blocking transcription.

Question 9

Trans-acting small RNAs (sRNAs):

Answer 9

• Can regulate translation initiation positively or negatively
• sRNA binds to mRNA inhibiting formation of secondary structures, allowing ribosome to initiate at SD/AUG
• or Preventing translation initiation at SD/AUG

Question 10

Osmolarity response - involve an sRNA

Answer 10

• The regulator allows one gene to be positively regulated while the other gene is effectively negatively regulated.
• small RNA response

Question 11

OmpF

Answer 11

a protein that forms pores in the membrane

Question 12

OmpC

Answer 12

A protein that forms pores in the membrane

Question 13

ompF:

Answer 13

• A porin gene

Question 14

OmpR:

Answer 14

• positively regulates ompC
• positively regulates the small RNA gene, micF
• Inhibits ompF expression

Question 15

micF

Answer 15

• A negative regulator of ompF

- A small RNA gene, which binds to ompF mRNA inhibitin translation

Question 16

Regulation of translation termination eg) Translation release factor 2 (RF2) regulates its own expression:

Answer 16

• Excess RF2: ribosomes terminate RF2 translation at codon 26, and expression doesn’t occur
• Limiting RF2: ribosomes do not terminate RF2 translation, ribosomes shift reading frame, due to an internal SD sequence that repositions the ribosome by 1 base, to translate full ORF

Question 17

How does density effect bacterial gene expression?

Answer 17

• Cells can uniformly change in some way by activating expression of response genes
• A signal produced reaches a critical threshold which activates a synchronised response

Question 18

Examples of this:

Answer 18

• Disease causing bacteria expressing disease causing genes only once they reach a certain level of population density
• Biofilms

Question 19

1. The trp operon is regulated by an attenuation mechanism
2. Some trpR- mutants are dominant (ie: trpR-D)
3. Tryptophan is the effector of the trpR regulatory protein
4. Transcription termination involves the formation of a stem-and-loop in the RNA

Answer 19

The trp operator is a region of dyad symmetry. Explain: 2 gives the best reasoning

2. TrpR is a dimer, so a mutant monomer can interfere with the function of a WT monomer and these are generally mutations in the DNA binding domain.
3. Region of dyad symmetry, but nothing todo with the operator or the binding of TrpR

Question 20

Unable to undergo lysis or form a lysogen (lethal mutation)

Answer 20

N mutant: because N is needed to progress the intermediate early genes, and if this is mutated it is lethal

Question 21

Unable to establish a lysogen

Answer 21

cI mutant: because lambda repressor is inactive and PR and PL cannot be repressed

Question 22

Unable to undergo lysis

Answer 22

Q mutant: unable to expressed late genes
Xis: could undergo lysis, could undergo lysogeny, could integrate, but wouldn’t be able to excise, so it can’t fully enter the lytic cycle

Question 23

Unable to both undergo lysis OR form a lysogen but lysogen cannot be induced to undergo lytic cycle

Answer 23

nutL mutant: required for cIII expression, more likely to undergo lysis, possibly unable to establish a lysogen

Question 24

More likely to form a lysogen than a WT lambda

Answer 24

OR3 mutant: overlaps PRM, so it is involved in Cro binding to inhibit cI expression, so more likely to get lysogeny
PRE mutant: repressor establishment, mutation here means it would be similar to a cII mutant, unable to form a lysogen