Week 10 - Regulation of Translation Flashcards
what initiates translation?
ribosome through first AUG codon
Bacterial translation
- transcription and translation are coupled
- polysomes begin assembly on elongating mRNA
Eukaryotic translation
- U protein complex is the primary message and can be spliced
- RNAP2 transcribing the gene
- mature mRNA transported into cytoplasm where ribosome goes onto mRNA and actively translates it
shine dalgarno sequence –
8 bases upstream from AUG seq.
Where does regulation of translation in eukaryotes occur?
cytoplasm
Molecules required for initiation of translation in bacteria
- 70s ribosome
- 30s ribosome and 16S RNA
- 50S ribosome –> not as important as 30 and 70s
- IF1 Initiation factor 1
- IF2
- IF3
- Initiation fMet-tRNAfmet (f-methionine tRNA)
Bacterial translation
- ribosome falls off mRNA –> will dissociate into 30s and 50s subunits when IF1 and IF3 bind to 30s
- fMet-tRNAfmet, mRNA and IF2 will bind to create 30s preinitiation complex (AUG AND ANTI-CODON HAVE NOT ALIGNED ATP)
- Initiation tRNA interacting with AUG = 30s initiation complex
- IF1 and IF3 are ejected = 30S RNA
- GTP in IF2 is hydrolyzed such that IF2 dissociates
- 50S subunit binds to 30s = 70s initiation complex (ready and poised for elongation of polypeptide chain)
what is important for placing mRNA onto ribosome and finding first AUG?
anti-SD and SD
- Watson and crick bp between these seq –> allows initiating tRNA to find first AUG
How is rate of translation regulated in Bacteria?
** reduce rate of initiation**
- inhibiting or enhancing the presentation of a ribosome BS
- SD seq or similar seq + AUG allow ribosome to recognized this as an initiation site of translation
- inhibiting the mRNA from entering the ribosome
- mRNA can bind but not enter ribosome channels
- 1st initiating contact RNA makes contact with AUG
What 4 factors influence the formation of secondary RNA structures?
- Binding of Proteins
- Binding of metabolites
- Binding of RNA
- Temperature
Stem loop (SL) secondary structures
- SD seq and AUG are hidden here such that no initiation complex form and ribosome cannot find SD or AUG
Binding of Proteins
- proteins binds to its mRNA to stabilize
- SD and AUG are in SL therefore no available for ribosome to bind
- S15 binds to mRNA expressed from gene required fro synthesis of this ribosomal protein
+/- Feedback loop: as S15 protein increases, its own synthesis is inhibited
when is S15 expressed?
When IPTG inhibits lac repressor form binding to lac operator
when are there high beta-galactosidase levels expressed?
-IPTG and therefore - S15
- no S15 to inhibit beta-galac.
What mutation would result in high expression in presence of IPTG?
- mutation in S15 such that structure is destabilized
- mutation in stem loop structure such that beta-gala. expression is high when +/- S15
riboswitch
senses levels of metabolites
TPP
thiamine pyrophosphate
what does TPP do?
regulates the level of translation of mRNA that encodes genes important for thiamine biosynthesis
what occurs to mRNA when little TPP?
mRNA will form secondary structure and expose SD and AUG to 30s ribosome –> where the initiation complex can form
What occurs when TPP binds to mRNA?
- induces another secondary structure to high SD and AUG from ribosome
- SD and AUG in stem loop –> initiation is SUPRESSED
what can metabolites influence?
expression of gene by binding to secondary RNA structure
Positive riboswitch
metabolite exposes SD and AUG so ribosome can bind
Negative riboswitch
another secondary structure induced to high AUG and SD in hairpin
Hfq
RNA chaperone protein that stabilizes structures between 2 RNAs (RNA negative regulation)
Hfq binds to ____ end of RNA
5’
RNA positive regulation
SD and AUG hidden in stem loop, when Hfq binds, RNA stimulates translation of another mRNA to expose SD and AUG –> ribsosome can now bind to initate translation
thermosensor
RNA structures at 5’ end of bacterial mRNAs that can cause different secondary structure to form
lower temp = SD and AUG expose
higher temp = SD and AUG placed in stem loop
when RNAP has gone through 3’ UTR, translation is ____ because SD and AUG bp with ___, hiding them
suppressed, 5’ end
5’ Cap and polyA tail
present in eukaryotic mRNA
Eukaryotic first AUG rule
initiation complex (40s) binds to mRNA, scans mRNA for first AUG, once found initiation of translation is set up, packaging of mRNA/ activation binds to 43s pre initiation complex where it is attached to mRNA, 5’ to 3’ scanning for first AUG, hydrolysis of ELF2-bound GTP and Pi release = 48S initiation complex
packaging of mRNA
pre-requisite of translation
elF4F complex
must bind to 5’ cap
- forms structure on 5’ cap
- structure binds to PolyA BP which creates circle so 5’ and 3’ ends are close in proximity
IRES (internal ribosome entry site)
-required for translation of internal cistrons
- RNA genome has multiple cistrons that need to be translated
