Peter Pristas. Translation

Transcription

1 Peter Pristas BNK1 Translation

2 The Central Dogma of Molecular Biology Adaptor molecule - trna

3 Deciphering the code in vivo Correlating a codon on mrna with an amino acid in a proteins requires 2 levels of specificity Correct amino acid must be attached to correct trna trna must recognize correct codons

4 Deciphering the genetic code 1960 s Nirenberg used cell-free extracts to translate artificial RNAs and deduced genetic code.

5 Genetic code only strand of DNA is transcribed into mrna the mrna is read in three s (codons) and translated by the ribosome into protein each codon only encodes one amino (no ambiguity) one amino may be encoded by more than one codon (redundancy) variability is in the 3rd codon position (wobble base) special codons are AUG -> start and methionine UAA/UGA/UAG -> stop

6 Genetics trna and rrna 7 different operonsin Escherichia coli rrna,rrnb,rrnc, rrnd,rrne, rrnf,rrng and rrng Each operon contains (i) one copy of the 23S, 16S and 5S rrnagenes (ii) 1-4 copiestrnagenes Post-transcriptional Regulation RNA Processing by RNAases RNaseIII, M5, M6 and M23

7 Processing of E. coli 30S pre-rrna The precursor RNA yields 23S, 16S and 5S rrnas as well as two different trnas. Other trnas are made from different precursor RNAs. Maturation of E. coli trna Tyr from its transcript

8 Generalized trna structure The positions of invariant and rarely varied bases are shown in purple. Regions in the D loop and the variable loop that can contain different numbers of nucleotides are shown in blue. The anticodon is shown in orange.

9 t RNA adaptor molecule

10 Examples of t-rna modifications Specific trna modifying enzymes

11 The trna anticodon loop trnas often recognize more than one codon due to wobble at the 3 rd base of a codon nonstandard base pairing.

12 Degeneracy: an organized feature of the genetic code Base pairing between mrna and trna is variable at the 3 rd position, and subjected to wobble

13 Charging trna ATP Each enzyme must recognize both the trna specific for an amino acid and the corresponding amino acid. Aminoacyl acid ATP-dependent and results in the cleavage of two high-energy phosphate bonds: Activated aa is trasferred to the trna with the relase of AMP Aminoacyladenylate Most trna synthetases must be able to recognize more than one type of trna (i.e. 6 codons for Arg). cognate trnas 3 aminoacyl-trna

14 Aminoacyl-tRNA synthetases They all carry out very similar tasks, but they vary greatly in size ( kdalton). 2 classes - structure of the active site Class I enzymes Generally (though not always) monomeric Attach the carboxyl of their target amino acid to the 2' OH of adenosine 76 in the trna molecule. Class II enzymes Generally dimericor tetrameric Attach their amino acid to the 3' OH of their trna Exception: Phe-tRNA synthetasewhich uses the 2' OH.

15 Aminoacyl-tRNA synthetases Activation occurs in two steps and is catalyzed by aminoacyltrnasynthetase: 1. The amino acid is covalently linked to AMP (acid anhydride linkage) 2. The activated amino acid is then transferred to its cognate trna, and AMP is released. Note that the reaction consumes one ATP. There is one aminoacyltrna synthetase for each amino acid. Because of the degeneracy of the genetic code, several trnas, all coding for the same amino acid, can be recognized by one aminoacyl trnasynthetase enzyme.

16 Proofreading The accuracy of charging trna with the proper amino acid is crucial because once charged, only the trna anticodon determines incorporation, NOT the attached amino acid. The error rate of charging is very low: 1 in 10,000. This is achieved by two means: The amino acid specificity pocket in a specificsynthetase will only bind amino acids similar in size and charge. Once a wrong aminoacyl-adenylate complex is formed (1st step), will hydrolyze the complex before it can be covalently attached to the trna (2nd step)

17 Class I AminoacyltRNA synthetases Class II

18 Prokaryotic and eukaryotic ribosomes differ in size and subunit structure. The size of the subunits is indicated by their sedimentation values (S). Ribosomes can be disassembled into protein and rrnasubunits. This process is reversible, i.e., fully functional ribosomes can be assembled in vitro from their proteins and rrnas.

19 Escherichia coli ribosome

20 Prokaryotes and Eukaryotes prokaryotes (bacteria) do not have nuclei eukaryotes segrege transcription in the nucleus. mrna is also preprocessed prior to translation in eukaryotes

21 Important facts on protein synthesis in both prokaryotes and eukaryotes Synthetase 1. Proteins are synthesized from amino- to carboxyl-terminus. 2. The mrna is read in the 5 to 3 direction. 3. All proteins start with methionine as the first amino acid. 4. There are two methionine-specific trnas: trna fmet and trna Met. 5. The amino group in Met-tRNA fmet is formylated in prokaryotes (on right). In eukaryotes, no formylation takes place. 6. Specific deformylases later remove the formylgroup from the protein. 7. Methionine-specific amino-peptidases cleave the terminal Met from some proteins but not all. N 10 -formyltetrahydrofolate Tetrahydrofolate Methionyl-tRNA (Met-tRNA fmet ) Transformylase Formylmethionyl-tRNAFmet (fmet-trna fmet )

22 Initiation of protein synthesis in prokaryotes 70S ribosome 1. IF1 and IF3 promote dissociation of a 70S ribosome. 2. IF2 binds to initiator trna together with GTP. 3. Together with the 30S ribosomal subunit this complex then attaches to a start codon that is preceded by a Shine-Dalgarno sequence. IF1 remains bound but IF3 is released. 4. The resulting 30S initiation complex is then joined by the larger 50S subunit. 5. This causes GTP to be hydrolyzed to GDP and P i, which initiates a conformational change in IF2. As a result of this change, IF2 is released together with IF1. 6. The 70S initiation complex is now ready for the next step of translation, elongation. The 50S subunit has three sites for trnabinding, called the P (petidyl) site, the A (aminoacyl) site, and the E (exit) site. The initiator trnais bound to the P site Initiator trna carried by IF2-GTP IF2 ready to bind GTP for another cycle + GDP + P i 50S released 30S subunit Shine-Dalgarno sequence Start codon (AUG or GUG or UUG) 30S initiation complex 50S subunit 70S initiation complex

23 Ribosomal binding site or Shine-Dalgarno sequence Polypurine sequence AGGAGG located on mrna just prior to an initiation codon Complementary to the sequence at the 3' end of 16S rrna Involved in binding of ribosome to mrna. Ribosomal protein L10 E. coli lacz Lambda phage cro

24 Multiple Shine-Dalgarno sequences in the polycistronic lac mrna A sequence in the 16S ribosomal RNA is complementary to the Shine-Dalgarno sequence.

25 Prokaryotic initiation factors Factor GTP Binding Role IF-1 No Blocks A site on 30S subunit so fmet-trnafbinds in P site IF-2 Yes Binds onlyfmet-trna (initiatortrna) IF-3 No Stabilizes 30S subunit dissociated from inactive 70S ribosome and aids mrna binding

26 Chain elongation in prokaryotic translation aa~trna 1. An aminoacyl trnain a complex with EF-Tuand GTP enters the A site of the ribosome. Upon codon-anticodonmatching, GTP is hydrolyzed and EF-Tu-GDP is released 2. A new peptide bond is formed by transfer of peptide chain from P site to aa-trnaat A site. The uncharged trna moves from the P site to the E site. 3. Translocation of peptidyl trna from A site to P site is catalyzed by EF-G. GTP is cleaved and the ribosome moves along mrna by one codon. 4. As soon as the A site is vacated the unchargetrnais released from the E site. GTP-aa~tRNA-EF-Tu complex P site E site Peptidyl trna Ribosome Cycle complete: ready to start again Codon 5 Codon 7 Codon 6 A site Regeneration of EF-Tu-GTP GDP 1 P i Binding of specific aa~trna to A site 3 2 Peptide bond formation; chain transfer from peptidyl trna to aminoacyl trna GDP + P i EF-Tu-GTP is regenerated in a separate cycle catalyzed by EF-Ts. Ribosome is ready to start another cycle trna

27 Prokaryotic elongation factors Factors GTP Binding Role EF-Tu Yes Binds all aminoacylated trnas (but not fmet-trnaf nor met-trnai) Most abundant protein in E. coli 1 copy per ribosome; 20,000 molecules per cell EF-Ts No Displaces GDP from EF-Tu EF-G Yes Translocation Only active when bound to GTP not GDP

28 Termination of translation in prokaryotes trna in P site carries completed polypeptide chain Stop codon Release factors RF1, RF2, RF3 1. trna in the P site carries a completed polypeptide chain. There is a stop codon in the mrna facing the A site. 2. RF1 or RF2 binds at or near the A site, RF3-GTP binds somewhere else. 3. Peptidyl transferase transfers the polypeptide chain from the P site to a water molecule. This releases the protein from ribosome. 4. The RF factors are then released, followed by the uncharged trna. 5. The ribosome is now unstable. Its instability is accentuated by the presence of the initiation factors IF1 and IF3. It dissociates into its subunits. Carboxyl end of chain is released upon hydrolysis of trna-peptide bond trna is released RF1 or RF2 binds at or near A site; RF3-GTP binds elsewher Ribosome dissociates. Probably the 50S subunit leaves first, stimulated by binding IF1 and IF3. The 30S subunit may either dissociate from the mrna or move to the next start codon nearby. 50S subunit 30S subunit

29 Prokaryotic termination factors TERMINATION CODONS UAA Ochre -most common UAG Amber UGA Opal No trna molecules to recognize Require release or termination factors Respond to termination codons 1 RF per ribosome Cause release of the completed polypeptide chain and the ribosome from mrna. Activate ribosome to hydrolyze Two: RF-1 and RF-2 RF-1 UAA and UAG RF-2 Dissociation of complex Release of peptide Expulsion oftrna UAA and UGA RF-3 RRF Releases the RF-1 and RF-2 from the complex (+GTP) Ribosome recycling factor to dissociate ribosomes

30 Translation Cycle mrna + 30S + 50S +fmet-trna mrna + 30S subunit + fmet-trna+ GTP + IF-1 + IF-2 + IF-3 Preinitiation Termination Initiation Elongation mrna + 70S ribosome +acyl-trna EF-Tu + EF-G + GTP mrna + 70S subunit + RF-1/RF-2 + RF-3 + RRF

31 Overview of transcription and translation process in prokaryotes

32 Transcription and translation are coupled in prokaryotes

33 Key points of translation in eukaryotes 1. The requirement for translation factors is considerably more complex than in prokaryotes. At least 11 proteins are required. 2. Some of the factors bind to mrna rather than the ribosome. 3. The major initiation factor eif2 forms a complex with Met-tRNA F and GTP. After binding to the 60S subunit, and formation of the 80S ribosome, eif2-gdp is recycled as in prokaryotes. 4. The initiator Met-tRNA F is not formylated. 5. There is no Shine-Dalgarno sequence. Instead the 40S subunit binds to the cap and scans the mrna until it finds the first AUG. This requires ATP. 6. Elongation is very similar to the prokaryotic process. 7. Termination is very similar to the prokaryotic process, but requires only a single release factor.

34 Translation initiation in eukaryotes 1. Pre-initiation complex forms small ribosomal subunit (40S) + eifs + trna 2. mrna bound via 7-meG-cap at 5 end 3. Ribosome scans for start codon, 4. Large subunit (60S) binds

35 Eukaryotic initiation factors Factor eif-1a eif-2 eif-2a eif-2b eif-3 eif-3a eif-4f eif-4a eif-4b eif-5 eif-5a Role binds and stimulates 43S complex formation bindsmet-trna, regulates ternary complex formation binding of ternary and 43S complex GTP to GDP conversion ribosome subunit dissociation and stabilizes 40Ssubunit ribosome subunit dissociation and stabilizes 60S subunit recognition and binding of 7-meG-cap at 5 mrna end helicase, mrna binding with 40S pre-initiation complex stimulation of eif-4a activity releasing of initiation factors from pre-initiation complex stimulation of first peptide bond formation

36 Translation and translocation are coupled in eukaryotes

37 Organelles have DNA

38 Translation in mitochondria - prokaryotic type of ribosomes - dependent upon proteins synthetised in cytoplasm (amino-acyltrna synthetases) Differences in genetic code used Mitochondria UGA AUA AGA/G Mammals Trp Met Stop Yeasts Trp Met Arg Drosophila Trp Met Ser Standard Stop Ile Arg

39 Translation in mitochondria

40 Post-translational modifications Removal of N-terminal Met in all eukaryotic proteins and in some prokaryotic acetylation of N-terminus common in eukaryots but not in prokaryots proteolytic processing of polypeptides, cleavage and protein splicing modifications of amino acids glycosylation, phosphorylation, lipidation non-covalent prosthetic groups: heme, etc.

41 Processing of preproteins

42 Covalent modification of proteins

43 Protein modification

44 Protein folding - chaperons

45 Protein degradation ubiquitin system