Lecture 10 - Ribosomes and Translation

tRNA

first sequence was reported in 1965

73-93 nucleotides, synthesized by RNAP III with type 2 internal promoters

final molecules are highly processed from primary RNA transcripts (requires successive cleavage and ligation reactions, many unusual base mods)

yeast has 272 nuclear tRNA genes, 59 are interrupted (single intron)

• no consensus sequence to be recognized by splicing enzymes

• splicing depends on recognition of common secondary structure in RNA

plants amphibians, animals also have interrupted tRNA genes

tRNA Modifications after Synthesis

D- dihydrouridine

i- inosine

T- thymidine

Ψ - pseudouridine

m - methyl group

are essential for stability, function, and structure of tRNA

tRNA Synthesis and Splicing

are heavily modified with 1% of the yeast genome codes for factors involved in tRNA processing and mod

1. 5’ leader sequence removed by RNase P (highly conserved)

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 (all bases can be modified by other enzymes)

Bacterial vs. Eukaryotic RNase P

bacterial - ribozyme

eukaryotic - nucleolar RNP enzyme

Conversion of mRNA into a Protein

tRNA is linked to a particular aa and it recognizes a codon in mRNA so that the corresponding aa can be added

• amino acids may have multiple codons but one tRNA has ONE unique tRNA

plants and animals have more tRNAs than bacteria - several different tRNAs for most amino acids

each tRNA is recognized by 1 of 20 enzymes - aminoacyl synthetase is first level of specificity in amino acid choice (one enzyme will attach one of 20 aa-charging tRNA)

• after amino acid is attached by charged tRNA, tRNA can recognize codon in mRNA and bring to growing polypeptide chain

• anticodon: part of tRNA that does specific interaction with codon in mRNA, a stretch of 3 sequential nucleotides

• tRNA function is dependent on 3D structure

tRNA Charging

aminoacyl-tRNA synthetase (ARS) catalyzes attachment of amino acid to free 2’ or 3’ OH of the ribose of the adenosine in the 3’ end of tRNA

• amino acid and adenosine monophosphate (AMP) are combined on ARS

• transferring of aminoacyl group from the enzyme complex to the tRNA ‘activates’ the amino acid residue → charged tRNA

• there are different enzymes based on if amino acid is added to 2’ or 3’ OH

• 20 different synthetases (one per amino acid)

• ribosome recognizes tRNA NOT the carrying amino acid (fidelity of the aminoacyl tRNA synthetase is crucial

Which part of the tRNA is the acceptor stem?

at the 3’ end where CCA is added as a step in tRNA processing

Steps of tRNA Charging

1. adenylylation of amino acid (uses ATP and releases inorganic phosphate PPi)

2. transfer of the adenylylated amino acid to 2’ or 3’ OH of the ribose of the A on (CCA at the 3’ end of tRNA) - charged tRNA

Non-Standard Base Pairing

cells have around 50 tRNAs for 20 amino acids

single tRNA can recognize more than one codon corresponding to an amino acid due to non-standard pairing in ‘wobble positions’

• antiparallel interaction - codon of mRNA is 5’-3’ and anticodon of tRNA is 3’-5’

• 1st nucleotide from codon is recognized by 3rd nucleotide from anticodon and vice versa

Great variability in recognition of ____ nucleotide in mRNA by ___ position in tRNA.

3rd - wobble nucleotide; 1st - wobble position

Aminoacyl tRNA Synthetase

ARS for arginine, leucine, and serine have to recognize 6 different tRNAs with different anticodons

has to select the proper amino acid to attach to the tRNA using the anticodon as the determinant

Ribosome

complex of rRNAs and proteins that directs elongation of a polypeptide (protein) - 2 subunits

synthesizes 17-21 aa/sec in prokaryotes, 6-9 amino acids/sec in eukaryotes

• subunits of ribosome are designated in Svedburg units (S), named after inventor of ultracentrifuge

• revealed differences between prokaryotes and eukaryotes’ ribosomes

What is S in Svedburg units?

a measure of sedimentation rate (velocity) of suspended particles when centrifuged under constant conditions - depends on size and shape of the particle

good measure of relative size is one is comparing same types of molecules (bigger S = faster sedimentation velocity)

Prokaryotic vs. Eukaryotic Ribosome

both have 5S rRNA subunit

prokaryotes have 16S rRNA subunit vs. 18S rRNA in eukaryotes

50S + 30S → prokaryotic 70S ribosome

60S + 40S → eukaryotic 80S ribosome

3 tRNA Binding Sites

assembled ribosome has APE sites

• aminoacyl site: incoming aminoacylated tRNAs bind

• peptidyl site: growing polypeptide chain is usually here where peptydyl-tRNA is

• exit side: exiting (empty) tRNA site

each site is located in the cleft of the small subunit, and has adjacent mRNA codons that are translated into amino acids

Stages of Translation

3 stages:

• initiation: assembly of a complete ribosome on an mRNA molecule at a correct point

• elongation: repeated cycles of amino acid addition

• termination: release of the new protein chain

Initiation

protein synthesis starts with initiator tRNA correctly positioned at start codon (AUG - Met)

euks and proks have 2 types of Met tRNAs charged with the same enzyme - methionyl tRNA synthetase

• tRNAMet is for methionine at internal codons of growing polypeptide (internal AUGs)

• tRNA iMet is for initiation where bacteria have modification-formyl-methionine

initiator tRNA allows for more regulation of initiation - alternate starts need to be recognized in bacteria

initial Met often removed during or immediately following translation

Prokaryotic Initiation of Translation

there is a conserved sequence in mRNA 8-13 nucleotides upstream from the first codon to be translated - ribosome binding site

• Shine-Delgarno 5’ - AGGAGGU - 3’

it base pairs with complementary sequence at 3’ end of 16S rRNA in the small 30S subunit that interacts with Shine-Delgarno

positions the ribosome correctly upstream from the initiation codon

Steps of Prokaryotic Translation

1. IF3 binds to free 30S subunit to prevent 50S subunit from binding

2. IF1 binds to prevent potential binding of tRNA to A site (amino acid landing)

3. IF2 is a GTPase that complexes with GTP and binds

4. mRNA binds to 30S through interaction of Shine-Dalgarno sequence with 16S rRNA by complementary bps

5. initiator tRNA binds by codon-anticodon base pairing to P site

6. this is the 30 initiation complex

7. 50S subunit binds

8. this displaces IF1 and IF3; GTP is hydrolyzed and IF2 is released

9. this is 70S initiation complex - ready to begin elongation

Eukaryotic Initiation of Translation

40S ribosomal subunit first binds initiator tRNA then 40S subunit-initiator tRNA complex binds mRNA and scans along mRNA until it reaches the right AUG and positions initiator tRNA there

first AUG ha to be in correct context where the optimal sequence context is Kozak consensus sequence

Steps of Eukaryotic Initiation

1. free 40S subunit complexes with eIF3 (large protein) and eIF1A to keep it apart from 60S subunit

2. ternary complex forms: initiator tRNA, eIF2 (GTPase), and GTP

3. ternary complex binds with 40S along with eIF1 → 43S preinitiation complex

4. on the other side, different eIF4 factors are involved in recognition of 5’ methyl cap and keep mRNA free of secondary structures using ATP E

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

6. 43S complex scans mRNA to find the right AUG inside the Kozak sequence by ATP hydrolysis energy

7. when Kozak sequence is found, eIF5 binds and displaces ALL other factors including eIF-GDP → 40S initiation complex is formed

8. 60S subunit binds by GTP hydrolysis

9. 80S initiation complex is formed

eIF2-GDP complex is recycled by ______ and converted back into ____ and ____.

eIF2B; eIF2 and GDP

GTP hydrolysis happens before large subunit binds in both prokaryotes and eukaryotes

Control of Beginning of Translation

eukaryotic cells have evolved elaborate mechanisms for translational control because of:

• changes in nutrient availability (mostly amino acids)

• changes in available cellular energy

• response to various forms of stress

• hormones and growth factor stimuli

control through:

1. phosphorylation of translation factors

2. multiple AUG codons in 5’ UTR

3. internal ribosome entry site (IRES)

where multiple AUGs and IRES frequently act together

In some mRNAs, inhibitory secondary structure in the ___ ____ impair efficient scanning of the ___ ____ ___ for the AUG start codon.

5’ untranslated region (UTR); small ribosomal subunit

Phosphorylation of Translation Factors

eIF2 is usually recycled by eIF2B with GTP and makes the ternary complex with tRNAMet BUT translation initiation of some genes can be down-regulated by phosphorylation of eIF2

1. amino acid starvation (and others) leads to accumulation of uncharged tRNAs

2. this activated protein kinase that will phosphorylate eIF2 to make it inactive

3. phosphorylated eIF2 cannot be recycled by eIF2B

this means when amino acid level is low, translation of some genes will slow or shut down

Multiple AUG Codons

some viral mRNA that use host translational machinery don’t have 5’ methyl caps but they have very long 5’ UTRs

multiple AUGs in their 5’ UTRs (also have IRESes)

two general types of multiple AUGs:

1. if Kozak sequence is very weak

2. AUG/UGA combo

Weak Kozak Sequence

ribosomal subunit scanning for AUG is then called ‘leaky scanning’ → results in upstream ORF

• ribosomal subunit moves to the second or third AUG (usually in frame)

• resulting proteins have different N-terminus which is the part that usually encodes a signal sequence (responsible for cell destination)

AUG/UGA Combo

short open reading frame between 5’ end of mRNA and beginning of the main coding sequence - upstream open reading frame (uORF)

• coded polypeptide is usually not functional

this stalls/decreases the translation of the actual downstream gene by “trapping” that scanning ribosome and causing it to drop down from the mRNA, before it reaches the AUG of the main protein coding sequence

basically, it makes a small peptide to stall the translation of the protein of interest since it takes energy and resources

• GCN4

• AA starvation

GCN4

type of AUG/UGA combo, is yeast amino acid synthesis genes

normal growth: lots of 43S preinitiation complexes; translation from uORGs

• the ribosomes that terminate translation on uORFs cannot resume scanning

• reinititation is very low from ‘real’ AUG5

• low level translation of GCN4

AA Starvation

type of AUG/UGA combo where fewer 43S preinitiation complexes formed by phosphorylation of eIF2

• more initiation from the ‘real’ AUG5

• more GCN4 is produced (translation factor)

• high levels of translation of amino acid biosynthetic genes

Features of 5’ UTR

they determine the impact of an uORF on the translation of the main ORF

sequence context makes it possible for the formation of RNA secondary structures

1. the length of the sequence upstream, and the sequence context of the uAUG both influence the ‘visibility’ of the start codon (hides the right AUG)

2. length of uORF and the sequence context of the uORF stop codon influence the competence of ribosomes to reinitiate on the main ORF once they have terminated translation (affects efficiency of assembly at main ORF)

3. uORF-encoded peptide can cause ligand-dependent stalling of the ribosome

4. sequences downstream of the uORF stop codon can influence both reinitiation and the stability of the mRNA

Internal Ribosome Entry Sites (IRES)

in the 5’ UTR

enables cap-dependent translation - bypass requirement for binding of eIF4E (binds methyl cap) and eIF4G (binds PABP) factors

is around 450 nucleotides long with complex secondary structure; rich in Us and Cs

43S preinitiation complex binds to IRES upstream from the ‘normal’ AUG and scans downstream from there

• IRES-specific binding proteins are required

• locates AUG in Kozak for initiation

Why is IRES important?

• viral infections: cap-dependent translation can be inhibited in cells due to cleavage of eIF4G (an adaptor to bridge the 40S subunit) by viral protease → outcome is only viral mRNA is translated

• mitosis: decreases cap-dependent translation

• cell in stress conditions: inhibits cap-dependent translation since translation factors get phosphorylated and turned off

• some cellular mRNAS have IRES AND cap

  • coding for some of stress induced proteins, some homeodomains proteins, some growth factors and apoptosis-associated proteins

  • their translation is not inhibited under conditions when overall (cap-dependent) protein synthesis is temporarily shut down

  • selective activation of IRES mediated translation

basically controls gene expression through cap-dependent translation, regulation of balance between apoptosis and cell division by translating a different sets of proteins

Review of Protein Coding Gene, mRNA, and ORF

this makes coding sequence

Generic Structure of Eukaryotic mRNA

Cytoplasmic Polyadenylation Element (CPE)

found in the 3’ UTR of some mRNA (usually maternal), works in the cytoplasm

is the signal for the length of the poly-A tail to be longer or shorter

different from poly-adenylation signal (PAS) which affects cleavage and poly-adenylation in the nucleus

Prokaryotic Elongation

after intitiation, tRNAi Met is on P site

1. amino-acyl-tRNA couples with elongation factor thermo-unstable (EF-Tu) and GTP into ternary complex, binds to A site on ribosome and pairs with codon on mRNA. this is catalyzed by GTP

2. peptide bond forms between previous amino acid and new one by peptidyl transferase reaction-previous amino acid is transferred to the amino acid carried by amino-acyl-tRNA which now becomes peptidyl-tRNA still at A site; unloaded tRNA is still at the P site

3. ribosome moves 3’ to move another codon into the A site (translocation is catalyzed by EF G (Pro) using GTP energy

   1. peptidyl-tRNA shifts to P site, empty tRNA is now at E site, A site is empty so new amino acid can come in

4. new amino-acyl-tRNA with GTP, EF-Tu, binds to A site in a GTP-dependent manner

EF-G During Elongation

small subunit head is twisted to facilitate codon translocation

Eukaryotic Elongation

1. amino-acyl-tRNA, eEF1A-eEF1B and GTP binds to A site

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

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

Puromycin

causes premature translation termination by acting as an aminoacyl-tRNA analog that is incorporated into the growing polypeptide chain - no carboxyl group available for peptide bond to form with amide from previous amino acid

Peptidyl Transferase

catalyzes the transfer of the growing peptide chain to the incoming activated amino acid and makes the peptide bond

tRNA makes direct contact with both 16S and 23S rRNAs in ribosome, and rRNAs are highly conserved way more than ribosomal proteins

ribosomal peptidyltransferase center is for: peptide bond formation and nascent peptide release during elongation and termination

Termination

there is no tRNA at stop codons (UGA, UAA, UAG)

the stop codon in the A site is recognized by the release factors

• with no charged tRNA, nascent peptide gets released once the ester bond linking the polypeptide to the P-site tRNA is hydrolyzed

• ribosome can be disassociated and recycled