21. Ribosomes and Translation

tRNA

• 73-93 nucleotides

• synthesized by RNAP III

• final molecules are highly processed from primary NR Atranscriits

steps in processing tRNA

1. 5’ leader sequence removed by RNase P

2. 3’ trailer sequence removed by endonuclease and exonuclease

3. CCA added to the 3’ end by nucleotidyl transferase

4. intron is spliced out by multiple enzymes

5. additional modifications at multiple residues

bacterial RNase P is a

ribozyme

eukaryotic RNase P is

nucleolar RNP enzyme

tRNA two functions

1. to be linked to a particular amino acid

2. to recognize a codon in mRNA so that the corresponding aa could be added

tRNA charging

1. CCA at 3’ end of tRNA = acceptor stem

2. aminoacyl-tRNA synthetase catalyzes attachment of aa to free 2’ or 3’ hydroyl of the ribose of the adenosine of the 3’ end of tRNA

3. AA and AMP combined on ARS

4. transferring of aminoacyl group from the enzyme complex to the tRNA "activates” the aa residue → charged tRNA

ribosome recognizes tRNA - NOT

the carrying aa - fideleity of the aminoacyl tRNA synthetase

charged tRNA

transfer of the adenylylated amino acid to 2’ or 3’ OH of the ribose of the A

cells have around

50 tRNAs for 20 aaa

single tRNA is able to recognize more than one codon

corresponding to an amino acid due to non-standard pairing in “wobble positions”

s is a measure of sedimentation rate (velocity) of suspended particles when centrifuged under constant conditions

velocity depends on both size and shape of the particle

• good measure of relative size if one is comparing same types of molecules (larger S value - faster sedimentation velocity)

mechanism of protein synthesis in three stages

1. initiation - the assembly of a complete ribosome on a mRNA molecule at a correct point

2. elongation - repeated cycles of amino acid addition

3. termination - release of the new protein chain

protein synthesis begins with

initiator tRNA correctly positioned at start codon (AUG)

eukaryotes and prokaryotes have two types of met tRNAs charged with the same enzyme (amino-acyl) methionyl tRNA synthetase

1. tRNAmet for methionine at internal codons of growing polypeptide

2. tRNAimet for initation (bacteria have a modification - formyl-methionine)

initiator tRNA allows for more regulation of translation initation

alternate starts (AUG and GUG) have to be recognized in bacteria

initial methionine often removed

during or immediately following translation

prokaryotic initiation of translation

1. shine dalgarno (AGGAGGU) sequence upstream from the first codon

2. base pairs with the complementary sequence at 3’ end of 16S rRNA in the small 30S subunit

3. positions the ribosome correctly upstream form the initiation codon

prokaryotic translation

1. IF3 binds to free 30s subunit

2. IF1 binds

3. IF2 (GTPase) complexes with GTP and binds

4. mRNA binds to 30s subunit through interaction of shine-dalgarno sequence with 16s RNA

5. initiator tRNA binds to P site → forms 30S initiation complex

6. 50S subunit binds

7. this displaces IF1 and IF3; GTP is hydrolyzed

   • energy consuming step - IF2 is released

8. this is 70S initiation complex

READY TO BEGIN ELONGATION

eukaryotic initiation of translation

1. 40 ribosomal subunit first binds initiator tRNA: then 40S subunit-initiator tRNA complex binds mRNA and scans along mRNA until it reaches an appropriate AUG and positions initiator tRNA there

   • this first aug has to be in correct sequence context - optimal kozak consensus sequence

2. free 40S subunit complexes with eIF3 and eIF1A

3. ternary complex forms = initiator tRNA, eIF2 and GTP

4. ternary complex binds to 40S along with EIF1 = 43S preinitiation complex

5. mRNA will bind to 43S complex through the 5’ methyl cap

in eukary translation, different eIF4 factors are involved in recognition of 5’methyl cap they keep

mRNA free of any secondary structures using the energy from ATP

in some mRNAs - inhibitory secondary structures in the 5’ untranslated region impair efficient scanning of the small ribosome subunit for the start AUG codon control through

1. phosphorylation of translation factors

2. multiple AUG codons in 5’ UTR

3. IRES (Internal Ribosome Entry site)

role of eIF2 in translation initiation

eIF2 binds GTP and tRNA met to form the ternary complex needed for translation initiation. it is recycled from GDP→ GTP by eIF2B

how does phophorylation of eIF2 regulate translation

phosphorylation prevents eIF2 from being recylced by eIF2B, blocking ternary complex formation and slowing or stopping translation

what triggers eIF2 phosphorylation during stress

amino acid starvation leads to accumulation of uncharged tRNAs

• this activates rotein kinase that will phophorylate eIF2

• phosphorylated eIF2 cannot be recycled by eIF2B

OUTCOME: when aa low, translation of some genes is going to slow or shut down

some viral mRNA

• lack 5’ methyl cap and they have very long 5’UTRs

• multiple AUGs in thier 5’ UTRs ( also have IREs)

• some cellular mRNA have it as well

if kozak sequence is very “weak” the scanning of the ribosomal subunits for the AUG is called leaky scanning

• resulting proteins differ in their N-terminus = part that usually encodes a singal sequence (responsible for cell destination)

purpose of multiple AUG codons

• to stall/decrease (down-regulate) the translation of the actual downstream gene by trapping the scanning ribosome and causing it to drop down from the mRNA (before reaching the AUG of the main protien coding)

Internal Ribosome Entry Sites - IRES

enable cap-independent translation - bypass requirement for binding of eIF4E (binds methyl cap) and eI4G (binds PABP) factors

why is IRES important

• viral infections → only viral mRNA translated

• mitosis

• cell in stress conditions

• some cellular mRNAs

control of gene expression through cap-independent translation

regulation of balance between cell apoptosis and cell division by translating a different sets of proteins

elongation prokaryote

1. amino-acyl-tRNA couples with EF-tu in prokaryotes and GTP into ternary complex

2. binds to A site on ribosome and pairs with codon on mRNA

3. catalyzed by GTP

4. peptide bond forms between previous aa and new aa - peptidyl transferase reaction

5. ribosome moves 3’ to another codon into the A site, translocation is catalyzed by EF G (pro) using GTP

6. empty tRNA released from E site

elongation in eukaryote

1. amino-acyl-tRNA, eEF1A - eEG1B and GTP binds to A site

2. peptide bond forms peptidyl-tRNA: sitll at the A site, unloaded tRNA still at the P site

3. ribosome translocates 3’ catalyzed by eEF2 (EU) using GTP energy