mol bio - module 7

optimization of genetic code

• maximize silent changes —> in line with common mutational signatures

• common mutation types cause conservative/tolerated substitutions rather than radical ones

• resource allocation —> could have been a problem back in the day (based on nitrogen/carbon/oxygen availability)

• amount of tRNAs and their modifications —> affects translation rates of individual codons which can affect mRNA stability and protein folding

which tRNA codes for stop codon

NONE —> tRNA’s job is to bring in an AA, so lack of one would signal that translation is done

nonsense mutations

special because they can be suppressed by a trans-acting factor

• trans factor can suppress premature stop codon and allow for read-through

  • tRNA mutates to suppress and bring in an AA —> cell still needs to be able to tolerate this change though
    

CCA

universal for all tRNAs and the hydroxyl of A is used to add the AA (charge the tRNA)

modified bases in tRNAS can:

• change their codon specificity

• alter their stability

• control their interactions with charging enzymes

wobble base pairing

3rd codon (first base for anticodon) can expand or contract interaction potential

how many tRNAs needed for MET

2!

1 for initial start and another for regular methionine

wobble base pairing vs watson crick

WC (optimal codons) is much faster than wobble

non-optimal codon synergy

cause ribosomes to slow down a lot

codon choice can allow for proper protein folding

ex) fast sequence with optimal codons can result in faster translation —> followed by less optimal codons to slow down translation a little so that previous segment can fold properly before moving on to the rest

codon choice controls mRNA stability

faster translation = better codon choice = mRNA more stable because we need more of it (positive feedback)

poor translation = mRNA unstable = negative feedback

• essentially, translation can act as an amplifier

optimal codons

most expressed tRNAs, WC base pairing

non-optimal codons

least expressed tRNAs, Wobble base pairing

slow decoding = unstable mRNA

E-site can act as a good timer

• nonoptimal codon = waiting for tRNA longer so A-site is empty

• tRNA in P-site eventually shifts to E-site and leaves

• if A-site is still empty when this happens, NOT5 recognizes this and binds to empty E-site

• recruits Ccr4 which is a deadenylase that stimulates decapping for mRNA, resulting in mRNA degradation

most RNA in cells

rRNA and then tRNA

most highly transcribed genes are:

ribosomal proteins

ribosomes structures

mostly RNA with some ribosomal proteins decorating the surface

only tRNA that binds to P site

initiator tRNA —> ribosome assembles around this and then closes P-site off

does ribosome check with AA is brought in along with tRNA?

NO!

ribosome only checks the tRNA

EfTu

elongation factor needed for each round of elongation

• GTPase that delivers tRNA to A-site and then hydrolyzes to induce conformational change —> if tRNA stays stable after this change, then it leaves the tRNA in the A-site and goes away

when is AA checked?

only at charging step with aminoacyltransferase

mRNA surveillance pathways

• nonsense mediated decay

• non-stop decay

• no-go decay

nonsense mediated decay

• as ribosome translates, it strips off the proteins on the mRNA

• if there’s an early stop codon, ribosome will fail to strip of EJC which then acts as timer and will result in mRNA degradation because it knows that the protein product is truncated

  • EJC is correlated to ORF to some extent

non-stop decay

• truncated mRNAs - no stop codon, message just ends

• causes ribosome to just stall at the end which also triggers decay

no-go decay

colliding ribosomes due to stalled ribosome in front → signal for a major problem

reasons for ribosome stalling

• secondary structure in mRNA it has to unwind first

• poly-Arginine codons because they can get stuck in channel due to biochemical properties

• inhibitory codon pair (synergize)

• P-site proline codon → structure is annoying

• poly-Lysine codons

• depurination of mRNA

recognition of stalled ribosome

collided ribosomes!

• collided ribosome recruits collision sensors that mark lead ribosome with Ub (K63-linked poly Ub)

• this recruits RQC complex to dissociate lead ribosome

• listerin then comes in and targets nascent polypeptide on lead ribosome through K48-linked polyUb to mark for degradation

pathway to recognize stalled monosome

• A-site empty for too long → Mag2 adds a monoUb

• results in polyUb (K63-linked)

• ribosome splits and small subunit is eliminated

full pathway for collided ribosomes

• collision sensed and ribosome is ubiquitinated

  • factors sent to inhibit initiation and activate decay

• RQC activated —> focused locally on specific ribosome

• when RQC is overwhelmed

  • signalling through kinases activates ISR which results in a global shut down of translation by inhibiting eIF2 (adds inhibitory phosphate)

    • OR if ISR is overwhelmed —> apoptosis

causes of collided ribosomes

RNA damage or formaldehyde (crosslink between proteins and RNA)

ribosomes might be better sensors of NA damage

SUPER dense on RNA and have less area to surveil (compared to the entire genome for DNA/RNA Polymerase)

value of sequencing RNA to learn about ribosomes

use Ribo-seq to find where ribosomes area and at what density

• comparing ribosome footprints to mRNA by RNA-seq can give us an idea about translation efficiency

• purification of different factors = specific kinds of ribosomes?

• discover unknown proteins by finding ribosome association in places we thought was noncoding

• find evidence for non-canonical start codon initiation, translation read through of stop codons, ribosome frameshifting

harringtonine

traps ribosomes on initiation codons —> determine non-canonical start codons

2 populations of fragment length from Ribo-seq

28 nt and 21 nt

large fragment (28 nt)

non-rotated state

pre-decoding → waiting for t-RNA in A site and GTP hydrolysis hasn’t occurred yet

small fragment (21 nt)

rotated state

post-decoding but pre-translocation → t-RNA in A-site but hasn’t shifted to P-site yet (GTP hydrolysis complete)