mrna and Genetic code standard

Transcription

1 Synthesis and Processing of the Proteome. (mrna and Genetic code: standard and variations; codon anti codon interactions), The role of trna in protein synthesis, Role of Ribosome in Protein synthesis (Ribosome structure, The players... mrna, trna, activating enzymes, initiation of translation in bacteria and eukaryotes, elongation of polypeptide in bacteria and eukaryotes and termination of polypepetide synthesis in bacteria and eukayrotes), Post translation modifications (Protein folding, Proteolysis, Protein chemical modification) Protein trafficking and degradation, inhibition of protein synthesis (antibiotic). Mitesh Shrestha

2 mrna and Genetic code standard

3 The Genetic Code Codons: 3 base code for the production of a specific amino acid, sequence of three of the four different nucleotides Since there are 4 bases and 3 positions in each codon, there are 4 x 4 x 4 = 64 possible codons 64 codons but only 20 amino acids, therefore most have more than 1 codon 3 of the 64 codons are used as STOP signals; they are found at the end of every gene and mark the end of the protein One codon is used as a START signal: it is at the start of every protein Universal: in all living organisms

4 Variations In 1979, researchers studying human mitochondrial genes discovered they used an alternative code Small variants such as translation of the codon UGA as tryptophan in Mycoplasma species, and translation of CUG as a serine rather than a leucine in yeasts of the "CTG clade" (Candida albicans is member of this group). In bacteria and archaea, GUG and UUG are common start codons, but in rare cases, certain proteins may use alternative start codons not normally used by that species. In certain proteins, non-standard amino acids are substituted for standard stop codons, depending on associated signal sequences in the messenger RNA. For example, UGA can code for selenocysteine and UAG can code for pyrrolysine. Selenocysteine is now viewed as the 21st amino acid, and pyrrolysine is viewed as the 22nd.

5 The Vertebrate Mitochondrial Code: Differences from the Standard Code: Codes Standard AGA Ter * Arg R AGG Ter * Arg R AUA Met M Ile I UGA Trp W Ter *

6 The Yeast Mitochondrial Code Differences from the Standard Code: Codes Standard AUA Met M Ile I CUU Thr T Leu L CUC Thr T Leu L CUA Thr T Leu L CUG Thr T Leu L UGA Trp W Ter * CGA absent Arg R CGC absent Arg R

7 trnas nt in length Structure of trna The acceptor arm is formed by seven base pairs between the 5 and 3 ends of the molecule. The amino acid is attached to the extreme 3 end of the trna, to the adenosine of the invariant CCA terminal sequence. The D arm, named after the modified nucleoside dihydrouridine, which is always present in this structure. The anticodon arm contains the triplet of nucleotides called the anticodon which base-pair with the mrna during translation. The V loop contains 3 5 nucleotides in Class 1 trnas or nucleotides in Class 2 trnas. The TΨC arm, named after the sequence thymidine-pseudouridine-cytosine, which is always present.

8 Structure of trna

9 Structure of trna The cloverleaf structure can be formed by virtually all trnas, the main exceptions being the trnas used in vertebrate mitochondria, which are coded by the mitochondrial genome and which sometimes lack parts of the structure. An example is the human mitochondrial trna Ser, which has no D arm. As well as the conserved secondary structure, the identities of nucleotides at some positions are completely invariant (always the same nucleotide) or semi-invariant (always a purine or always a pyrimidine), and the positions of the modified nucleotides are almost always the same.

10 The role of trna in protein synthesis Transfer RNAs play the central role in translation. Adaptor molecules, which form the link between the mrna and the polypeptide that is being synthesized. A physical link, trnas binding to both the mrna and the growing polypeptide, and an informational link, trnas ensuring that the polypeptide being synthesized has the amino acid sequence that is denoted, via the genetic code, by the sequence of nucleotides in the mrna. trnas play this dual role through aminoacylation, the process by which the correct amino acid is attached to each trna, and codon-anticodon recognition, the interaction between trna and mrna.

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12 Aminoacylation Process of adding an aminoacyl group to a compound. It produces a trna molecule with its CCA 3' end covalently linked to an amino acid. Each trna is aminoacylated (or charged) with a specific amino acid by an aminoacyl trna synthetase. There is normally a single aminoacyl trna synthetase for each amino acid, despite the fact that there can be more than one trna, and more than one anticodon, for an amino acid. Recognition of the appropriate trna by the synthetases is not mediated solely by the anticodon, and the acceptor stem often plays a prominent role. As only 20 amino acids are designated by the genetic code, this means that all organisms have at least some isoaccepting trnas, different trnas that are specific for the same amino acid. The terminology used when describing trnas is to indicate the amino acid specificity with a superscript suffix, using the numbers 1, 2, etc., to distinguish different isoacceptors: for example, two trnas specific for glycine would be written as trna Gly1 and trna Gly2.

13 Aminoacylation The attachment of amino acids to trnas - charging in molecular biology jargon - is the function of the group of enzymes called aminoacyl-trna synthetases. The chemical reaction that results in aminoacylation occurs in two steps. An activated amino acid intermediate is first formed by reaction between the amino acid and ATP, and then the amino acid is transferred to the 3 end of the trna, the link being formed between the -COOH group of the amino acid and the -OH group attached to either the 2 or 3 carbon on the sugar of the last nucleotide, which is always an A

14 Aminoacylation The result of aminoacylation by a Class II aminoacyl-trna synthetase is shown, the amino acid being attached via its -COOH group to the 3 -OH of the terminal nucleotide of the trna. A Class I aminoacyl-trna synthetase attaches the amino acid to the 2 -OH group.

15 Features of aminoacyl-trna synthetases Feature Class I enzymes Class II enzymes Structure of the enzyme active site Interaction with the trna Orientation of the bound trna Amino acid attachment Enzymes for * Parallel β-sheet Minor groove of the acceptor stem V loop faces away from the enzyme To the 2 -OH of the terminal nucleotide of the trna Arg, Cys, Gln, Glu, Ile, Leu, Lys1, Met, Trp, Tyr, Val Antiparallel β-sheet Major groove of the acceptor stem V loop faces the enzyme To the 3 -OH of the terminal nucleotide of the trna Ala, Asn, Asp, Gly, His, LysII, Phe, Pro, Thr, Ser

16 Aminoacylation With a few exceptions, organisms have 20 aminoacyltrna synthetases, one for each amino acid. This means that groups of isoaccepting trnas are aminoacylated by a single enzyme. Although the basic chemical reaction is the same for each amino acid, the 20 aminoacyl-trna synthetases fall into two distinct groups, Class I and Class II, with several important differences between them. In particular, Class I enzymes attach the amino acid to the 2 -OH group of the terminal nucleotide of the trna, whereas Class II enzymes attach the amino acid to the 3 -OH group

17 Building a Molecule of trna A specific enzyme called an aminoacyl-trna synthetase joins each amino acid to the correct trna Amino acid Aminoacyl-tRNA synthetase (enzyme) P P P ATP Adenosine 1 Active site binds the amino acid and ATP. 2 ATP loses two P groups and joins amino acid as AMP. Pyrophosphate P P i P Adenosine 3 Appropriate trna covalently Bonds to amino Acid, displacing AMP. Phosphates P i P i trna P Adenosine AMP 4 Activated amino acid is released by the enzyme. Aminoacyl trna (an activated amino acid )

18 Unusual types of aminoacylation

19 Codon Anti codon interactions Codon anticodon pairing is unstable in the absence of the ribosome.

20 Codon Anti codon interactions A straightforward process involving base-pairing between the anticodon of the trna and a codon in the mrna

21 Wobble hypothesis Francis Crick The first two bases in the codon create the coding specificity, for they form strong Watson-Crick base pairs and bond strongly to the anticodon of the trna. When reading 5' to 3, the first nucleotide in the anticodon (which is on the trna and pairs with the last nucleotide of the codon on the mrna) determines how many nucleotides the trna actually distinguishes. If the first nucleotide in the anticodon is a C or an A pairing is specific and acknowledges original Watson-Crick pairing, that is only one specific codon can be paired to that trna. If the first nucleotide is U or G, the pairing is less specific and in fact two bases can be interchangeably recognized by the trna. Inosine displays the true qualities of wobble, in that if that is the first nucleotide in the anticodon then any of three bases in the original codon can be matched with the trna. Due to the specificity inherent in the first two nucleotides of the codon, if one amino acid codes for multiple anticodons and those anticodons differ in either the second or third position (first or second position in the codon) then a different trna is required for that anticodon. The minimum requirement to satisfy all possible codons (61 excluding three stop codons) is 32 trnas. That is 31 trna's for the amino acids and one initiation codon.

22 Wobble hypothesis

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24 Codon Anti codon interactions The specificity of aminoacylation ensures that the trna carries the amino acid denoted by the codon that it pairs with, and the ribosome controls the topology of the interaction in such a way that only a single triplet of nucleotides is available for pairing.

25 Role of the Ribosome in Protein Synthesis Two active roles in protein synthesis Ribosomes coordinate protein synthesis by placing the mrna, aminoacyl-trnas and associated protein factors in their correct positions relative to one another. Components of ribosomes, including the rrnas, catalyze at least some of the chemical reactions occurring during translation.

26 Structure of Ribosome

27 Ribosome The ribosome has three binding sites for trna The P site The A site The E site P site (Peptidyl-tRNA binding site) E site (Exit site) E P A A site (AminoacyltRNA binding site) Large subunit mrna binding site Small subunit (b) Schematic model showing binding sites. A ribosome has an mrna binding site and three trna binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams.

28 Ribosome in protein synthesis. In E. coli, ribosome is assembled on mrna at initiation codon w/translation initiation factor IF-3 (prevents premature dissociation); 3 of 16S rrna attached to ribosome binding site (Shine-Dalgarno sequence).

29 Translation The process in which cellular ribosomes create proteins. Translation proceeds in three phases: Initiation: The ribosome assembles around the target mrna. The first trna is attached at the start codon. Elongation: The trna transfers an amino acid to the trna corresponding to the next codon. The ribosome then moves (translocates) to the next mrna codon to continue the process, creating an amino acid chain. Termination: When a stop codon is reached, the ribosome releases the polypeptide.

30 BACTERIAL TRANSLATION Factor Function Initiation factors IF-1 Unclear; X-ray crystallography studies show that binding of IF-1 blocks the A site (see page 329), so its function may be to prevent premature entry of trnas into the A site. Alternatively IF-1 may cause conformational changes that prepare the small subunit for attachment to the large subunit IF-2 IF-3 Elongation factors EF-Tu EF-Ts EF-G Release factors RF-1 RF-2 RF-3 Directs the initiator trnai Met to its correct position in the initiation complex Prevents premature reassociation of the large and small subunits of the ribosome Directs the next trna to its correct position in the ribosome Regenerates EF-Tu after the latter has yielded the energy contained in its attached GTP molecule Mediates translocation Recognizes the termination codons 5 -UAA-3 and 5 -UAG-3 Recognizes 5 -UAA-3 and 5 -UGA-3 Stimulates dissociation of RF1 and RF2 from the ribosome after termination Ribosome recycling factor RRF Responsible for disassociating the ribosome subunits after translation has terminated

31 Initiation of translation The main difference between initiation of translation in bacteria and eukaryotes is that in bacteria the translation initiation complex is built up directly at the initiation codon, the point at which protein synthesis will begin, whereas eukaryotes use a more indirect process for locating the initiation point.

32 Initiation of Translation The initiation stage of translation brings together mrna, trna bearing the first amino acid of the polypeptide, and two subunits of a ribosome 3 5 U A C A U G 5 3 P site Large ribosomal subunit Initiator trna mrna GTP GDP E A 5 Start codon mrna binding site Small ribosomal subunit Translation initiation complex 1 A small ribosomal subunit binds to a molecule of mrna. In a prokaryotic cell, the mrna binding site on this subunit recognizes a specific nucleotide sequence on the mrna just upstream of the start codon. An initiator trna, with the anticodon UAC, base-pairs with the start codon, AUG. This trna carries the amino acid methionine (Met). 2 The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiation factors (not shown) are required to bring all the translation components together. GTP provides the energy for the assembly. The initiator trna is in the P site; the A site is available to the trna bearing the next amino acid.

33 Initiation of protein synthesis in E. coli requires initiation factors IF-1, IF-2, & IF-3. IF-3 binds to the 30S ribosomal subunit, freeing it from its complex with the 50S subunit. IF-1 assists binding of IF-3 to the 30S ribosomal subunit. IF-1 also occludes the A site of the small ribosomal subunit, helping insure that the initiation aa-trna fmet-trna fmet can bind only in the P site & that no other aa-trna can bind in the A site during initiation. IF-2 is a small GTP-binding protein. IF-2-GTP binds the initiator fmet-trna fmet & helps it to dock with the small ribosome subunit.

34 As mrna binds, IF-3 helps to correctly position the complex such that the trna fmet interacts via base pairing with the mrna initiation codon (AUG). A region of mrna upstream of the initiation codon, the Shine-Dalgarno sequence, base pairs with the 3' end of the 16S rrna. This positions the 30S ribosomal subunit in relation to the initiation codon. As the large ribosomal subunit joins the complex, GTP on IF-2 is hydrolyzed, leading to dissociation of IF-2-GDP and dissociation of IF-1. A domain of the large ribosomal subunit serves as GAP (GTPase activating protein) for IF-2. Once the two ribosomal subunits come together, the mrna is threaded through a curved channel that wraps around the "neck" region of the small subunit.

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36 Initiation of translation in eukaryotes Initiation of protein synthesis is much more complex in eukaryotes, & requires a large number of protein factors. Some eukaryotic initiation factors (e.g., eif3 & eif4g) serve as scaffolds, with multiple domains that bind other proteins during assembly of large initiation complexes.

37 Eukaryotic translation factors Factor Function Initiation factors eif-1 Component of the pre-initiation complex eif-1a Component of the pre-initiation complex eif-2 Binds to the initiator trna Met within the ternary complex component of the pre-initiation complex; phosphorylation of eif-2 results in a global repression of translation eif-3 Component of the pre-initiation complex; makes direct contact with eif-4g and so forms the link with the cap binding complex eif-4a eif-4b eif-4e eif-4f eif-4g eif-5 eif-6 Component of the cap binding complex; a helicase that aids scanning by breaking intramolecular base pairs in the mrna Aids scanning, possibly by acting as a helicase that breaks intramolecular base pairs in the mrna Component of the cap binding complex, possibly the component that makes direct contact with the cap structure at the 5 end of the mrna The cap binding complex, comprising eif-4a, eif-4e and eif-4g, which makes the primary contact with the cap structure at the 5 end of the mrna Component of the cap binding complex; forms a bridge between the cap binding complex and eif-3 in the pre-initiation complex; in at least some organisms, eif-4g also forms an association with the poly(a) tail, via the polyadenylate-binding protein Aids release of the other initiation factors at the completion of initiation Associated with the large subunit of the ribosome; prevents large subunits from attaching to small subunits in the cytoplasm Elongation factors eef-1 Complex of four subunits (eef-1a, eef-1b, eef-1d and eef-1g); directs the next trna to its correct position in the ribosome eef-2 Mediates translocation Release factors erf-1 Recognizes the termination codon erf-3 Possibly stimulates dissociation of erf-1 from the ribosome after termination; possibly causes the ribosome subunits to disassociate after termination of translation

38 Initiation of translation in eukaryotes The first step involves assembly of the pre-initiation complex. This structure comprises the 40S subunit of the ribosome, a ternary complex made up of the initiation factor eif-2 bound to the initiator trnamet and a molecule of GTP, and three additional initiation factors, eif- 1, eif-1a, and eif-3. As in bacteria, the initiator trna is distinct from the normal trnamet that recognizes internal 5 -AUG-3 codons but, unlike bacteria, it is aminoacylated with normal methionine, not the formylated version.

39 Initiation of translation in eukaryotes After assembly, the pre-initiation complex associates with the 5 end of the mrna. This step requires the cap binding complex (sometimes called eif-4f), which comprises the initiation factors eif-4a, eif-4e and eif-4g. The contact with the cap might be made by eif-4e alone or might involve a more general interaction with the cap binding complex. The factor eif-4g acts as a bridge between eif-4e, bound to the cap, and eif-3, attached to the pre-initiation complex. The result is that the pre-initiation complex becomes attached to the 5 region of the mrna. Attachment of the pre-initiation complex to the mrna is also influenced by the poly(a) tail, at the distant 3 end of the mrna. This interaction is thought to be mediated by the polyadenylate-binding protein (PADP), which is attached to the poly(a) tail. In yeast and plants it has been shown that PADP can form an association with eif-4g, this association requiring that the mrna bends back on itself. With artificially uncapped mrnas, the PADP interaction is sufficient to load the pre-initiation complex onto the 5 end of the mrna, but under normal circumstances the cap structure and poly(a) tail probably work together. The poly(a) tail could have an important regulatory role, as the length of the tail appears to be correlated with the extent of initiation that occurs with a particular mrna.

40 Initiation of translation in eukaryotes After becoming attached to the 5 end of the mrna, the initiation complex, as it is now called, has to scan along the molecule and find the initiation codon. The leader regions of eukaryotic mrnas can be several tens, or even hundreds, of nucleotides in length and often contain regions that form hairpins and other base-paired structures. These are probably removed by a combination of eif-4a and eif-4b. eif-4a, and possibly also eif-4b, has a helicase activity and so is able to break intramolecular base pairs in the mrna, freeing the passage for the initiation complex. The initiation codon, which is usually 5 -AUG-3 in eukaryotes, is recognizable because it is contained in a short consensus sequence, 5 -ACCAUGG-3, referred to as the Kozak consensus.

41 Initiation of translation in eukaryotes Once the initiation complex is positioned over the initiation codon, the large subunit of the ribosome attaches. As in bacteria, this requires hydrolysis of GTP and leads to release of the initiation factors. Two final initiation factors are involved at this stage: eif-5, which aids release of the other factors, and eif-6, which is associated with the unbound large subunit and prevents it from attaching to a small subunit in the cytoplasm.

42 Initiation of translation in eukaryotes

43 Initiation of translation in eukaryotes Translation initiation in eukaryote. Preinitiation complex scans along mrna until it reaches the initiation codon (a few tens or hundreds nt downstream & located within Kozak consensus sequence) large subunits then attach.

44 Initiation of eukaryotic translation without scanning The scanning system for initiation of translation does not apply to every eukaryotic mrna. This was first recognized with the picornaviruses, a group of viruses with RNA genomes which includes the human poliovirus and rhinovirus, the latter being responsible for the common cold. Transcripts from these viruses are not capped but instead have an internal ribosome entry site (IRES) which is similar in function to the ribosome binding site of bacteria, although the sequences of IRESs and their positions relative to the initiation codon are more variable than the bacterial versions. The presence of IRESs on their transcripts means that picornaviruses can block protein synthesis in the host cell by inactivating the cap binding complex, without affecting translation of their own transcripts, although this is not a normal part of the infection strategy of all picornaviruses. Examples are the mrnas for the mammalian immunoglobulin heavy-chain binding protein and the Drosophila Antennapedia protein

45 Usually a pre-initiation complex forms, including: several initiation factors the small ribosomal subunit the loaded initiator trna, Met-tRNA i Met. This then binds to a separate complex that includes: mrna initiation factors including ones that interact with the 5' methylguanosine cap & the 3' poly-a tail, structures unique to eukaryotic mrna. Within this complex mrna is thought to circularize via interactions between factors that associate with the 5' cap & with a poly-a binding protein..

46 After the initiation complex assembles, it translocates along the mrna in a process called scanning, until the initiation codon is reached. Scanning is facilitated by eukaryotic initiation factor eif4a, which functions as an ATP-dependent helicase to unwind mrna secondary structure while releasing bound proteins. A short sequence of bases adjacent to the AUG initiation codon may aid in recognition of the start site. After the initiation codon is recognized, there is hydrolysis of GTP and release of initiation factors, as the large ribosomal subunit joins the complex and elongation commences.

47 Eukaryotic Translation Translation of mrna is highly regulated in multi-cellular eukaryotic organisms, whereas in prokaryotes regulation occurs mainly at the level of transcription. There is global regulation of protein synthesis. E.g., protein synthesis may be regulated in relation to the cell cycle or in response to cellular stresses such as starvation or accumulation of unfolded proteins in the endoplasmic reticulum. Mechanisms include regulation by signal-activated phosphorylation or dephosphorylation of initiation and elongation factors.

48 Regulation of translation initiation. Global regulation (e.g. under stressful conditions) by eif-2 phosphorylation prevents GTP binding, therefore represses translation; Transcript specific regulation by feedback inhibition or feedback activation mechanisms (left) Figure 13.17a Genomes 3 ( Garland Science 2007)

49 Translation of particular mrnas may be inhibited by small single-stranded microrna molecules about nucleotides long. MicroRNAs bind via base-pairing to 3' un-translated regions of mrna along with a protein complex RISC (RNAinduced silencing complex), inhibiting translation and in some cases promoting mrna degradation. Tissue-specific expression of particular genomeencoded micrornas is an essential regulatory mechanism controlling embryonic development. Some forms of cancer are associated with altered expression of micrornas that regulate synthesis of proteins relevant to cell cycle progression or apoptosis.

50 Elongation of translation Attachment of the large subunit results in two sites at which aminoacyl-trnas can bind. The first of these, the P or peptidyl site, is already occupied by the initiator trna i Met, charged with N-formylmethionine or methionine, and base-paired with the initiation codon. The second site, the A or aminoacyl site, covers the second codon in the open reading frame

51 Elongation of translation

52 Elongation of translation The A site becomes filled with the appropriate aminoacyl-trna, which in E. coli is brought into position by the elongation factor EF-Tu, which ensures that only trnas that carry the correct amino acid are able to enter the ribosome, mischarged trnas being rejected at this point. EF-Tu is an example of a G protein, meaning that it binds a molecule of GTP which it can hydrolyze to release energy. In eukaryotes the equivalent factor is called eef-1, which is a complex of four subunits: eef-1a, eef-1b, eef-1d and eef-1g. The first of these exists in at least two forms, eef-1a1 and eef-1a2, which are highly similar proteins that probably have equivalent functions in different tissues. Specific contacts between the trna, mrna and the 16S rrna within the A site ensure that only the correct trna is accepted. These contacts are able to discriminate between a codon-anticodon interaction in which all three base pairs have formed, and one in which one or more mis-pairs are present, the latter signaling that the wrong trna is present. This is probably just one part of a series of safeguards that ensure the accuracy of the translation process

53 Elongation of translation When the aminoacyl-trna has entered the A site, a peptide bond is formed between the two amino acids. This involves a peptidyl transferase enzyme, which releases the amino acid from the initiator trnai Met and then forms a peptide bond between this amino acid and the one attached to the second trna. In bacteria, the peptidyl transferase activity resides in the 23S rrna of the large subunit, and so is an example of a ribozyme. The reaction is energy dependent and requires hydrolysis of the GTP attached to EF-Tu (eef-1 in eukaryotes). This inactivates EF-Tu, which is ejected from the ribosome and regenerated by EF-Ts. A eukaryotic equivalent of EF-Ts has not been identified, and it is possible that one of the subunits of eef-1 has the regenerative activity

54 P site trna A site trna Transpeptidation O O (peptide bond O P O CH Adenine O P O CH Adenine 2 2 O O formation) involves O H H O H H H H H H nucleophilic attack O OH O OH O C O C of the amino N of HC R HC R the amino acid NH : NH 2 linked to the 3'OH of the terminal O C adenosine of the HC R + NH trna in the A site 3 on the carbonyl C of the amino acid (with attached nascent polypeptide) in ester linkage to the trna in the P site. The reaction is promoted by the geometry of the active site consisting solely of residues of the 23S rrna of the large ribosomal subunit. No protein is found at the active site.

55 O trna O P O O CH 2 H H O O C HC P site NH Adenine O H H OH R O trna O P O O CH 2 O Adenine H H H O H OH O C HC R NH 2 : A site O C HC R NH 3 + The 23S rrna may be considered a "ribozyme." As part of the reaction a proton (H + ) is extracted from the attacking amino N.

56 O trna O P O O P site CH 2 O Adenine H H H OH H OH O trna O P O O A site CH 2 O Adenine H H H O H OH O C HC R NH O C HC NH R O C HC R NH 3 + This H + is then donated to the hydroxyl of the trna in the P site, as the ester linkage is cleaved.

57 It had been proposed that a ring N of a highly conserved adenosine at the active site might act as a catalyst mediating this H + transfer. However, on the basis of recent structural and mutational evidence it has been concluded that the active site adenine is essential only as part of the structure of the active site that positions the substrates correctly. H + shuttling is attributed instead to the adjacent ribose 2' hydroxyl group of the P-site peptidyl-trna, along with ribose hydroxyls of active site rrna residues & structured water molecules that collectively form a H-bonded network at the active site.

58 The nascent polypeptide, one residue longer, is now linked to the A-site trna. O trna O P O O P site CH 2 O Adenine H H H OH H OH O trna O P O A site O CH 2 O Adenine H H H O H OH O C HC R NH O C HC R NH O C HC R NH 3 +

59 Translocation Now the dipeptide corresponding to the first two codons in the open reading frame is attached to the trna in the A site. The next step is translocation, during which three things happen at once: The ribosome moves along three nucleotides, so the next codon enters the A site. The dipeptide-trna in the A site moves to the P site. The deacylated trna in the P site moves to a third position, the E or exit site, in bacteria or, in eukaryotes, is simply ejected from the ribosome.

60 Translocation Translocation requires hydrolysis of a molecule of GTP and is mediated by EF-G in bacteria and by eef-2 in eukaryotes. Electron microscopy of ribosomes at different intermediate stages in translocation suggests that the two subunits rotate slightly in opposite directions, opening up the space between them and enabling the ribosome to slide along the mrna. Translocation results in the A site becoming vacant, allowing a new aminoacyl-trna to enter. The elongation cycle is now repeated, and continues until the end of the open reading frame is reached.

61 trna grey, EF-Tu red, EF-G blue The unloaded trna in the P site will shift to the E (exit) site during translocation. Translocation of the ribosome relative to mrna involves the GTP-binding protein EF-G. The size & shape of EF-G are comparable to that of the complex of EF-Tu with an aa-trna. Structural studies & molecular dynamics indicate that EF-G-GTP binding in the vicinity of the A site causes a ratchet-like motion of the small ribosomal subunit against the large subunit.

62 large subunit trna EF-G small subunit mrna location Figure provided by Dr. J. Frank, Wadsworth Center. The trna with attached nascent polypeptide is pushed from the A site to the P site. Unloaded trna that was in the P site shifts to the E site. Since trnas are linked to mrna by codon-anticodon base pairing, the mrna moves relative to the ribosome.

63 Additionally, it has been postulated that translocation is spontaneous after peptide bond formation because: the deacylated trna in the P site has a higher affinity for the E site, & the peptidyl-trna in the A site has a higher affinity for the P site. Interaction with the ribosome, which acts as GAP (GTPase activating protein) for EF-G, causes EF-G to hydrolyze its bound GTP to GDP + P i. EF-G-GDP then dissociates from the ribosome. A domain of EF-G functions as its own GEF (guanine nucleotide exchange factor) to regenerate EF-G-GTP.

64 The continued codon-anticodon base paring of the trna in the E site is postulated to have a role in preventing potentially serious frame-shift errors, e.g., such as would occur if the trnas were to able to shift laterally by one base pair. Normally the empty trna is released from the E site after binding of the correct aminoacyl-trna at the causes a decreased affinity for trna in the E site. only A site

65 Translation: Elongation Elongation: the ribosome moves down the messenger RNA, adding new amino acids to the growing polypeptide chain. The ribosome has 2 sites for binding transfer RNA. The first RNA with its attached amino acid binds to the first site, and then the transfer RNA corresponding to the second codon bind to the second site.

66 Translation: Elongation The ribosome then removes the amino acid from the first transfer RNA and attaches it to the second amino acid. At this point, the first transfer RNA is empty: no attached amino acid, and the second transfer RNA has a chain of 2 amino acids attached to it.

67 Elongation of the Polypeptide Chain In the elongation stage, amino acids are added one by one to the preceding amino acid TRANSCRIPTION TRANSLATION DNA mrna Ribosome Polypeptide Ribosome ready for next aminoacyl trna Amino end of polypeptide mrna 5 E P A site site 2 3 GTP 1 Codon recognition. The anticodon of an incoming aminoacyl trna base-pairs with the complementary mrna codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. 2 GDP E E P A P A 3 Translocation. The ribosome translocates the trna in the A site to the P site. The empty trna in the P site is moved to the E site, where it is released. The mrna moves along with its bound trnas, bringing the next codon to be translated into the A site. GDP GTP E P A 2 Peptide bond formation. An rrna molecule of the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the trna in the A site.

68 Frameshifting and other unusual events during elongation The straightforward codon-by-codon translation of an mrna is looked upon as the standard way in which proteins are synthesized. But an increasing number of unusual elongation events are being discovered. One of these is frameshifting, which occurs when a ribosome pauses in the middle of an mrna, moves back one nucleotide or, less frequently, forward one nucleotide, and then continues translation. The result is that the codons that are read after the pause are not contiguous with the preceding set of codons: they lie in a different reading frame. Spontaneous frameshifts occur randomly and are deleterious because the polypeptide synthesized after the frameshift has the incorrect amino acid sequence. But not all frameshifts are spontaneous: a few mrnas utilize programmed frameshifting to induce the ribosome to change frame at a specific point within the transcript.

69 Programmed frameshifting Occurs in all types of organism, from bacteria through to humans, as well as during expression of a number of viral genomes. An example occurs during synthesis of DNA polymerase III in E. coli, the main enzyme involved in replication of DNA. Two of the DNA polymerase III subunits, γ and τ, are coded by a single gene, dnax. Subunit τ is the full-length translation product of the dnax mrna, and subunit γ is a shortened version. Synthesis of γ involves a frameshift in the middle of the dnax mrna, the ribosome encountering a termination codon immediately after the frameshift and so producing the truncated γ version of the translation product.

70 Programmed frameshifting It is thought that the frameshift is induced by three features of the dnax mrna: A hairpin loop, located immediately after the frameshift position, which stalls the ribosome; A sequence similar to a ribosome binding site immediately upstream of the frameshift position, which is thought to base-pair with the 16S rrna (as does an authentic ribosome binding site), again causing the ribosome to stall; The codon 5 -AAG-3 at the frameshift position. The presence of a modified nucleotide at the wobble position of the trna Lys that decodes 5 -AAG-3 means that the codonanticodon interaction is relatively weak at this position, enabling the frameshift to occur.

71 Translational Slippage Enables a single ribosome to translate an mrna that contains copies of two or more genes. This means that, for example, a single ribosome can synthesize each of the five proteins coded by the mrna transcribed from the tryptophan operon of E. coli. When the ribosome reaches the end of one series of codons it releases the protein it has just made, slips to the next initiation codon, and begins synthesizing the next protein.

72 Translational bypassing A form of slippage in which a large part of an mrna is skipped during translation, elongation of the original protein continuing after the bypassing event. The bypass starts and ends either at two identical codons or at two codons that can be translated by the same trna by wobble. This suggests that the jump is controlled by the trna attached to the growing polypeptide, which scans the mrna as the ribosome tracks along, and halts the bypass when a new codon to which it can base-pair is reached.

73 Translational bypassing Translational bypassing of 44 nucleotides occurs in E. coli during translation of the mrna for gene 60 of T4 bacteriophage, which codes for a DNA topoisomerase subunit. Similar events have also been identified in a variety of other bacteria. Bypassing could result in two different proteins being synthesized from one mrna - one protein from normal translation and one from bypassing - but whether this is its general function is not yet known.

74 Frameshifting and other unusual events during elongation

75 Polyribosomes A number of ribosomes can translate a single mrna molecule simultaneously forming a polyribosome Polyribosomes enable a cell to make many copies of a polypeptide very quickly (a) Incoming ribosomal subunits Start of mrna (5 end) Growing polypeptides End of mrna (3 end) Completed polypeptide An mrna molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mrna 0.1 µm This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

76 Termination of translation Protein synthesis ends when one of the three termination codons is reached. The A site is now entered not by a trna but by a protein release factor. Bacteria have three of these: RF-1 which recognizes the termination codons 5 -UAA-3 and 5 -UAG-3, RF-2 which recognizes 5 -UAA-3 and 5 -UGA-3, and RF-3 which stimulates release of RF1 and RF2 from the ribosome after termination, in a reaction requiring energy from the hydrolysis of GTP. Eukaryotes have just two release factors: erf-1, which recognizes the termination codon, and erf-3, which might play the same role as RF-3 although this has not been proven. The structure of erf-1 has been solved by X-ray crystallography, showing that the shape of this protein is very similar to that of a trna. This gives an indication of how the release factor is able to enter the A site when the termination codon is reached.

77 Termination of translation The release factors terminate translation but they do not appear to be responsible for disassociation of the ribosomal subunits, at least not in bacteria. This is the function of an additional protein called ribosome recycling factor (RRF) which, like erf-1, has a trna-like structure. RRF probably enters the P or A site and unlocks the ribosome. Disassociation requires energy, which is released from GTP by EF-G, one of the elongation factors, and also requires the initiation factor IF-3 to prevent the subunits from attaching together again. A eukaryotic equivalent of RRF has not been identified, and this may be one of the functions of erf-3. The disassociated ribosome subunits enter the cytoplasmic pool, where they remain until used again in another round of translation.

78 Termination of translation

79 Post-translational Processing of Proteins

80 Protein folding Four levels of protein structure; need correct tertiary structure to be activated; a dynamic process; for large proteins, renaturation is not always spontaneous due to (1) tendency to form insoluble aggregates; (2) more stable alternative folding pathways.

81 Protein folding Protein folding is assisted by molecular chaperons (to hold proteins in an open conformation for folding) & chaperonins (a protein complex to promote folding through a cavity & proofread incorrectly folded proteins into correct folding).

82 Proteolytic cleavage Protein cutting is either endprocessing (to cut off N or C terminals to make functional proteins) or polyprotein processing (to cut into small pieces of functional proteins).

83 Proteolytic cleavage An example of endprocessing is pre-pro-insulin. Step 1. Cut off 24 amino acids from N terminal to give pro--insulin; step 2. Cut internal B chain to give insulin.

84 Proteolytic cleavage An example of poly-protein processing used as a way to reduce size of genomes w/ a single gene & 1 promoter & 1 terminator; can be spliced in various ways in different cells.

85 Chemical modification

86 Chemical modification More complex modification is glycosylation used to add large carbohydrate side chains to Serine (O-linked) or Asparagine (N-linked).

87 Intein splicing A protein version of RNA splicing (vs. extein); first discovered in yeast in 1990; also found in bacteria & archaea; most ~150 aa & self-catalyzed; intein homing (convert a intein- gene into a intein+ gene; used as a mechanism to propagate).

88 Targeting Polypeptides to Specific Locations Completed proteins are targeted to specific sites in the cell Two populations of ribosomes are evident in cells: free ribsomes (in the cytosol) and bound ribosomes (attached to the ER) Free ribosomes mostly synthesize proteins that function in the cytosol Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell Ribosomes are identical and can switch from free to bound

89 Targeting Polypeptides to Specific Locations Polypeptide synthesis always begins in the cytosol Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER Polypeptides destined for the ER or for secretion are marked by a signal peptide A signal-recognition particle (SRP) binds to the signal peptide The SRP brings the signal peptide and its ribosome to the ER Ribosomes mrna Signal peptide Signalrecognition particle SRP (SRP) receptor protein CYTOSOL Signal peptide removed ER membrane Protein ER LUMEN Translocation complex

90 Protein Degradation The concept that the proteome of a cell can change over time requires not only de novo protein synthesis but also the removal of proteins whose functions are no longer required. This removal must be highly selective so that only the correct proteins are degraded, and must also be rapid in order to account for the abrupt changes that occur under certain conditions, for example during key transitions in the cell cycle

91 Protein Degradation A link between ubiquitin and protein degradation was first established in 1975 when it was shown that this abundant 76-amino-acid protein is involved in energy-dependent proteolysis reactions in rabbit cells Subsequent research identified a series of three enzymes that attach ubiquitin molecules, singly or in chains, to lysine amino acids in proteins that are targeted for breakdown.

92 Protein Degradation Whether or not a protein becomes ubiquitinated depends on the presence or absence within it of amino acid motifs that act as degradation-susceptibility signals. These signals have not been completely characterized but there are thought to be at least ten different types in S. cerevisiae, including: The N-degron, a sequence element present at the N terminus of a protein; PEST sequences, internal sequences that are rich in proline (P), glutamic acid (E), serine (S) and threonine (T). These sequences are permanent features of the proteins that contain them and so cannot be straightforward degradation signals : if they were then these proteins would be broken down as soon as they are synthesized. Instead, they must determine susceptibility to degradation and hence the general stability of a protein in the cell. How this might be linked to the controlled breakdown of selected proteins at specific times, for instance during the cell cycle, is not yet clear.

93 Protein Degradation The second component of the ubiquitin-dependent degradation pathway is the proteasome, the structure within which ubiquitinated proteins are broken down. In eukaryotes the proteasome is a large, multi-subunit structure with a sedimentation coefficient of 26S, comprising a hollow cylinder of 20S and two caps of 19S. Archaea also have proteasomes of about the same size but these are less complex, being composed of multiple copies of just two proteins; eukaryotic proteasomes contain 14 different types of protein subunit. The entrance into the cavity within the proteasome is narrow, and a protein must be unfolded before it can enter. This unfolding probably occurs through an energy-dependent process and may involve structures similar to chaperonins but with unfolding rather than folding activity. After unfolding, the protein can enter the proteasome within which it is cleaved into short peptides 4 10 amino acids in length. These are released back into the cytoplasm where they are broken down into individual amino acids which can be re-utilized in protein synthesis.

94

95 Protein Degradation

96 A summary of transcription and translation in a eukaryotic cell TRANSCRIPTION 1 RNA is transcribed from a DNA template. 3 DNA 5 RNA transcript RNA polymerase RNA PROCESSING Exon 2 In eukaryotes, the RNA transcript (premrna) is spliced and modified to produce mrna, which moves from the nucleus to the cytoplasm. NUCLEUS RNA transcript (pre-mrna) Intron Aminoacyl-tRNA synthetase CYTOPLASM Amino FORMATION OF acid INITIATION COMPLEX AMINO ACID ACTIVATION trna 3 After leaving the 4 Each amino acid nucleus, mrna attaches attaches to its proper trna to the ribosome. with the help of a specific enzyme and ATP. mrna Growing polypeptide Activated amino acid Ribosomal subunits 5 TRANSLATION Figure E A A A A U G G U U U A U G Ribosome Codon Anticodon 5 A succession of trnas add their amino acids to the polypeptide chain as the mrna is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.)