L31 Regulation of Translation in Prokaryotes

Question 1

Translation in Prokaryotes - Ribosome

Answer 1

see onenote diagram

Polyribosomal polysome

Question 2

mRNAs in prokaryotes - operons

Answer 2

mRNA arranged in operons

Each ORF usually contains a RBS

Question 3

Initiation of translation in prokaryotes

Answer 3

see onenote slides

1. translation initiation factors (IF)
   - IF prevents translation until initiation tRNA comes in, then IF dissociates and large ribosomal subunit associates
2. RBS recognition
3. association of 50S large subunit

Question 4

Regulating translation initiation in prokaryotes

Answer 4

see onenote

Question 5

Regulating translation termination in prokaryotes

Answer 5

see onenote

Internal RBS inducing translation frameshift

Question 6

General mechanism for regulating translation initiation - A&B

Answer 6

see onenote slides

A.
RNA binding protein (RBP) prevents translation initiation

why is it important that the RBP doesn’t bind to the RBS?

B.
secondary structure prevents translation of ORF1, translation of ORF2 disrupts structure allowing translation

Question 7

Translation regulation of ribosomal proteins

Answer 7

see onenote slides

ribosomal RNA and ribosomal proteins must be stoichiometrically co-regulated

ribosomal proteins arranged in operons

gene dosage experiments reveal autorepression of ribosomal protein operons

Question 8

How are rRNA and ribosomal proteins autorepressed? A&B

Answer 8

see onenote slides

A.
ribosomal proteins interact with rRNA to assist formation of secondary structures, if rRNA is in excess, proteins will be used

B.

• If ribosomal protein in excess, protein 2 binds to mRNA, preventing translation of protein 1.
• Translation of ORF2 inhibited by structure in mRNA

Question 9

Translation regulation of spc operon by ribosomal S8 protein

Answer 9

see onenote slides

S8 protein acts as translational repressor of L5 ORF (on the spc operon)

A.
S8 binds to form structure A in 16S rRNA

B.
If in excess, ribosomal protein S8 binds structure B in mRNA encoding S8, inhibiting translation of itself

S8 affinity for 16S > mRNA

Question 10

Riboswitches - can regulate transcription termination

Answer 10

see onenote

can regulate transcription termination

structure upstream of mRNA
When there’s excess metabolite, changes secondary structure => forms transcriptional terminator

Question 11

E.coli lysC riboswitch

Answer 11

see onenote slides

Regulates translation initiation

in mRNA upstream of lys transporter ORF and lys biosynthesis ORF

Question 12

Riboswitches - can regulate transcription initiation

Answer 12

structure upstream of mRNA

When there’s excess metabolite, changes secondary structure => forms sequestering helix => ribosome can no longer bind to RBS

Question 13

Riboswitches - can regulate transcription initiation and mRNA degradation

non-nucleolytic repression vs nucleolytic repression mechanism

Answer 13

see onenote slides

RNase E sites
- degradosome can access mRNA in OFF state when lys binds

Question 14

RNA thermosensor regulates translation in Listeria

Answer 14

see onenote

Listeria
- Switches between non-pathogenic and pathogenic state through translational regulation of the virulence gene

37 degrees
- prfA, a virulence protein required for host infection, RBS accessible at this temperature => prfA can be translated

Question 15

Listeria’s nucleotide mutation

Answer 15

mutation of nucleotide required for secondary mRNA structure abolishes thermosensing

Question 16

pfrA-UTR

Answer 16

see onenote diagram

can function as a thermoswitch in E.coli

Question 17

trans-acting sRNAs can regulation translation initiation +ve or -ve

Answer 17

see onenote

sRNA binds to mRNA:

a. inhibit formation of secondary structures, allows initiation
b. prevent translation initiation at RBS/AUG

Question 18

Osmolarity response

Answer 18

see onenote slides

involves sRNA

EnvZ - component 1
OmpR - component 2

OmpF and OmpC proteins form pores in membrane

DBD regulates porin gene expression

phosphorylation of OmpR activates ompC and inhibits ompF expression (via micF binding)

Question 19

Translation termination in prokaryotes requires RF1/RF2

Answer 19

see onenote

RF = release factors
RF prevents tRNA from coming in

Question 20

Regulation of translation termination by RF2

Answer 20

see onenote

excess RF2

limiting RF2 - When ribosome reaches this stop codon, it stalls as there is no RF2 to come in to terminate translation => frameshift occurs

RF2 regulates its own expression by translation termination

Question 21

Final thought of translation in prokaryotes

Answer 21

Important implications

• Problem in terms of antibiotic availability and resistance
• Large number of antibiotics are inhibitors of prokaryotic translations, not healthy for you (organelles translation is similar to prokaryotes)