Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Section Q Protein Synthesis Q1 Aspects of Protein Q2 Mechanism of Protein Synthesis Q3 Initiation in Eukaryotes Q4 Translation Control and Post-translational Events

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Q1 Aspects of Protein Codon-anticodon interaction Wobble Ribosome binding site Polysomes Initiator tRNA

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Codon-anticodon Interaction In the cleft of the ribosome, an anti-parallel formation of three base pairs occurs between the codon on the mRNA and the anticodon on the tRNA. The anticondon at one end of the tRNA interacts with a complementary triplet codon of bases on the mRNA, when both are brought together in the cleft of the ribosome. The interaction is anti-parallel in nature. Some highly purified tRNA molecules were found to interact with more than one codon, and this ability correlated with the presence of modified nucleosides in the 5’-anticodon position.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Wobble-I The wobble hypothesis was suggested by Francis Crick to explain the redundancy of genetic code. His specific predictions are shown in right table along with actual observation.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences non-standard base pairs The 5'-anticodon base is able to undergo more movement than the other two bases and can thus form non-standard base pairs as long as the distances between the ribose units are close to normal. To explain the redundancy of the genetic code. 18 aa are encoded by more than one triplet codons which usually differ at 5’-anticodin base. Wobble-II

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Base pairings at the wobble position Wobble-III I: inosine

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Only for prokaryotic translation A purine-rich sequence usually containing all or part of the sequence 5'- AGGAGGU-3' Locates 8-13 nt upstream of the initiation codon in prokaryotic mRNA Function: To position the ribosome binding for initiation of protein synthesis Ribosome binding site － Shine-Dalgarno sequence Ribosome mRNA Ribosome

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Ribosome binding site － Shine-Dalgarno sequence John Shine Shine-Dalgarno gene sequenceJohn Shine is known to most undergraduate biology students for his role in defining the Shine-Dalgarno gene sequence, which is responsible for the initiation and termination of protein-synthesis. Education 1972 BSc (Hons) 1st Class, Australian National University 1975 PhD, Australian National University 2006 DSc (Honoris causa) The University of New South

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Polysomes Each mRNA transcript is read by more than one ribosome. A second, third, fourth, etc. ribosome starts to read the mRNA transcript before the first ribosome has completed the synthesis of one polypeptide chain. polyribosomespolysomesMultiple ribosomes on a single mRNA transcript are called polyribosomes or polysomes. Multiple ribosomes can not be positioned closer than 80 nt. Polysomes

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Electron micrographs of ribosomes actively engaged in protein synthesis revealed by "beads on a string" appearance. Polysomes

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Initiator tRNA The initiator tRNA fMet-tRNA fMet fMet-tRNA fMet

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Q2 Mechanism of Protein Synthesis Overview Initiation Elongation Termination

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Overview Three stages: 1.Initiation 1.Initiation: the assembly of a ribosome on an mRNA molecule; 2.Elongation 2.Elongation: repeated cycles of amino acid addition; 3.Termination 3.Termination: the release of the new protein chain.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Initiation in E coli 123 G G 5’ 3’ 3 2 5’ 3’ 1 2 G 5’ 3’ 1 G 30S subunit Initiation factors fMet Initiator tRNA fMet 30S initiation Complex 50S subunit Shine-Dalgarno sequence AUG mRNA 70S initiation Complex A-siteP-site Initiation Steps 1. IF 1 and IF 3 bind to a free 30S subunit. 2. IF 2 complexed with GTP then binds to the small subunit. It will assist the charged initiation tRNA to bind. 3. The 30S subunit attaches to an mRNA molecule marking use of the ribosome-binding site (RBS) on the mRNA. 4. The initiator tRNA can then bind to the complex by base pairing of its anti-codon with the mRNA. 5. The 50S subunit can now bind, which displaces IF 1 and IF 2, and the GTP is hydrolyzed in this energy-consuming step S initiation complex formed at the end of this phase.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Elongation in E coli 5’ 3’ aa 5’ 3’ aa 5’ 3’ aa 3’ 5’ Empty A-site Binding of aa-tRNA to A-site aa Peptide bond formation From A- to P-site P-site Elongation Steps Aminoacyl-tRNA delivery 1. Aminoacyl-tRNA delivery. EF-Tu is required to delivery the aa-tRNA to A-site. Energy is from GTP. Peptide bond formation peptidyl transferase 2. Peptide bond formation. After aa- tRNA delivery, the A- and P-site are both occupied and the two amino acids that are joined closely. The peptidyl transferase activity of the 50S subunit can now form a peptide bond between these two amino acids. Translocation 3. Translocation. A complex of EF-G (translocase) and GTP binds to the ribosome and the discharged tRNA is elected from the P-site, the peptidyl-tRNA is moved from the A-site to P-site.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences EF-G:GDP EF-G:GTP EF-Tu:GTP Aa-tRNA-EF-Tu:GTP Aa-tRNA EF-Tu:GDP EF-Tu:EF-Ts EF-Ts GDP EF-Ts GTP

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Role of elongation factors in prokaryotes EF-Tu –It is a G protein (GTP/GDP-binding protein) –ActiveGTP –Active when GTP is bound (Kd = M) –InactiveGDP –Inactive with GDP is bound (Kd = M), slow offrate –Its off rate is increased by EF-Ts EF-Ts –It is a guanosine nucleotide exchange factor (GEF) for EF-Tu EF-G –It is a G protein –There is no known GEF for EF-G

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Release factor 1 or 2 Release factor 3 Release factor tRNA Termination There are no tRNA species that normally recognize stop codon. rf RF1UAA UAGRF2 UAAUGAInstead, protein factors called release factors interact with these codons. RF1 recognizes UAA and UAG, and RF2 recognizes UAA and UGA. RF3RF1RF2RF3 helps either RF1 or RF2 to carry out the reaction. The release factors mark peptidyl transferase transfer polypeptide to water rather than to the usual aminoacyl-tRNA, and thus the new protein is released. To remove the uncharged tRNA from P-site and release the mRNA, EF-G is needed. Termination in E coli UGA Termination codon UGA G 5’ 3’ aa N 5’ 3’ aa N G N Released polypeptide chain G Peptidyl transerase 5’ 3’

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Q3 Initiation in Eukaryotes Overview Scanning Initiation Elongation and Termination

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Overview A comparison of the factors involved in prokaryotes and eukaryotes is given in Table 1.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Differences between prokaryotes and eukaryotes Bacteria Ribosome: 30S+50S →70S Few initiation factors (three factors) –IF1, IF2, IF3 Elongation factors –EF1A (EF-Tu), EF1B (EF-Ts), EF2 (EF-G) Release factors –RF1, RF2, RF3 Ribosome recycling factor –RRF mRNA is not capped Direct binding of 30S particle next to initiation codon (AUG) at Shine- Dalgarno sequence, 5’-AGGAGGU-3’ Translation coupled to transcription Eukaryotes Ribosome: 40S+60S → 80S Many initiation factors (14 factors) –eIF1, eIF1A, eIF2, eIF2B, eIF3, eIF4A, eIF4B, eIF4E, eIF4F, eIF4G, eIF4H, eIF5, eIF5B, eIF6 Elongation factors –eEF1, eEF2 Release factors –eRF1 (or eRF2), eRF3 Most mRNA is capped at 5’ end and poly (A) at 3’ end 40S particle is recruited to 5’ cap structure or poly(A) tail or an internal ribosome entry site (IRES) Translation in always in cytoplasm apart from transcription

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Scanning Since there is no Shine-Dalgarno sequence in eukaryotic mRNA, the mechanism of selecting the start codon must be different. scanning hypothesis AUGProf. Kozak proposed a scanning hypothesis in which the 40S subunit, already containing initiator tRNA, attaches to the 5’- end of the mRNA and scans along the mRNA until it finds an appropriate AUG. 5’cap AUGEukaryotic small ribosome submit complex bind to the 5’cap region of the mRNA and moves along it scanning for an AUG start codon.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences 1. Dissociation of ribosome 4C binding eIF1A (4C) and eIF3 to 40S 2. Recruit ternary complex (fMet-tRNAi, eIF2, GTP) to 40S 3. Recruit mRNA to 43S → 46S particle AUG S scans mRNA to reach AUG 80S 5. Initiation factors leave and 80S joins 6. Elongation begins Initiation

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Initiation 1. Dissociation of ribosome binding of eIF4C-A-site eIF1A (eIF4C-A-site) and eIF3 to 40S eIF3 is a huge complex (MW=0.69 MDa) of 11 subunits not homologous to IF3 Possibly eIF5, eIF5B and eIF1 bind at this point as well eIF1 seems to play a similar role as IF3-C 2. Recruit ternary complex (fMet-tRNAi, eIF2, GTP) to 40S eIF2 is a complex of three subunits ( , ,  ) of 123 kDa GTP is loaded by eIF2B Regulated by phosphorylation 3. Recruit mRNA to 43S mRNA is bound to the eIF4F complex (4E, 4G, 4A) 4. Scan mRNA to reach AUG Scanning requires a defined number of eIFs 5. Initiation factors leave and 80S joins eIF5 is the exchange GEF for eIF2→ eIF2 release Ribosome is GEF for eIF5B→ eIF5B release 6. Elongation begins

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Elongation and Termination Elongation The protein synthesis elongation cycle in prokaryotes and eukaryotes is quite similar. –Three factors (EF-Tu, EF-Ts and EF-G) are required properties similar to their prokaryotic counterparts. –eEF1 α, eEF1 βγ and eEF2 have the roles described for EF-Tu, EF-Ts and EF-G respectively. Termination In eukaryotes, a single release factor, eRF, recognizes all three stop codons and performs the roles carried out by RF1 (or RF2) plus RF3 in prokaryotes. eRF requires GTP for activity, but it is not yet clear whether there is a eukaryotic equivalent of ribosome release factor required for dissociation of the subunits from the mRNA.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Q 4 Translation Control and Post-translational Events Translational control Poly-proteins Protein Targeting Protein modification Protein degradation

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Translational control In prokaryotes 1.Short antisense molecules can obscure ribosome binding; 2.The formation of stems and loops can inhibit exonuleases; 3.The relative stability to nucleases of parts of the polycistronic mRNA; 4.The binding of protein that prevent ribosome access. In eukaryotes 1.Generally control protein amount by transcription of genes; 2.The repeats of the sequence 5’-AUUUA-3’ can make the mRNA for rapid degradation and thus limited translation; 3.Protein binding can mask the mRNA (masked mRNA) and prevent translation.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Polyproteins (1) Bacteriophage and viral transcipts and (2) many mRNAs for hormones in eukaryotes (e.g. pro-opiomelanocortin) are translated to give a single polypeptide chain that is cleaved subsequently by specific proteases to produce multiple mature protein from one translation product. The parent polypeptide is called polyprotein.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Protein Targeting-I Signal sequenceSignal sequence –It is a short peptide sequences which composed of about 13~36 amino acids. signal sequence –Function: The signal sequence causes the translating ribosome to bind factors that make the ribosome dock with a membrane and transfer the protein through the membrane as it is synthesized. –Usually the signal sequence is then cleaved off by signal peptidase. Signal recognition particle (SRP)Signal recognition particle (SRP) –SRP can recognize ribosomes with signal peptide of the nascent chain. SRP receptor (docking protein)SRP receptor (docking protein) –SRP with the arrested ribosome can bind to SRP receptor on the cytosolic side of the Endoplasmic Reticulum (ER); Ribosome receptor proteinRibosome receptor protein –When the ribosome becomes attached to ribosome receptor protein on the ER, SRP is released and can be re-used. –The ribosome is able to continue translation, and the nascent polypeptide chain is pushed through into the lumen of the ER.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Protein Targeting-II GlycosylationGlycosylation –The protein in ER is usually modified by glycosylation, and different patterns of Glycosylation seem to control the finial location of the protein. Nuclear localization signal (nls)Nuclear localization signal (nls) nl s –Different N-terminal sequences can cause protein to be imported into mitochondria or chloroplasts, and the internal sequence –Lys-Lys-Lys- Arg-Lys, or any five continuous amino acids, can be a nuclear localization signal (nls). It cause the protein to be imported into the nucleus.

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences The secretory pathway in eukaryotes (co-translational targeting)

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Protein modification The most common alterations to nascent polypeptides are those of cleavage and chemical modification. (by amino- & carboxypeptideases) 1.Polypeptide cleavage (by amino- & carboxypeptideases) To remove signal peptides; To release mature fragments from polyprotein; To remove internal peptides as well as To trim both N- and C-termini. 2.Chemical modification: Acetylation; Hydroxylation; Phosphorylation; Methylation; Glycosylation. Phosphorylation

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences Protein degradation A protein that is damaged, modified or has an inherently destabilizing N-terminal residue becomes ubiquitinylated by covalent linkage of molecules of the small, highly conserved, ubiquitin, via its C-terminal Gly, to lysine residues in the protein. The ubiquitinylated protein is digested by a 26S protease complex in a reaction that requires ATP and releases intact ubiquitin for re-use. In eukaryotes, N-terminal residue plays a critical role in inherent stability: t 1/2 >20 hours: Ala, Cys, Gly, Met, Pro, Ser, Thr, Val (eight aa); t 1/2 2~30 min: Arg, His, Ile, Leu, Lys, Phe, Trp, Tyr (eight aa); Destabilizing: Asn, Asp, Gln, Glu. (4aa) 26S protease complex

Section O: RNA Processing and RNPs.Yang Xu, College of Life Sciences That’s all for Section Q