3D-structure of bacterial ribosomes, the machines that make proteins

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1 3D-structure of bacterial ribosomes, the machines that make proteins
FIGURE The bacterial ribosome. Our understanding of ribosome structure has been greatly enhanced by multiple high-resolution images of the bacterial ribosome and its subunits, contributed by several research groups. A sampling is presented here. (a) The 50S and 30S bacterial subunits, split apart to visualize the surfaces that interact in the active ribosome. The structure on the left is the 50S subunit (derived from PDB ID 2OW8, 1VSA, and 1GIX), with tRNAs (displayed as green backbone structures) bound to sites E, P, and A, described later in the text; the tRNA anticodons are in red. Proteins appear as blue wormlike structures representing the peptide backbone; the rRNA as a gray rendering of the surface features. The structure on the right is the 30S subunit (derived from PDB ID 2OW8). Protein backbones are brown wormlike structures and the rRNA is a lighter tan surface rendering. The part of the mRNA that interacts with the tRNA anticodons is shown in red. The rest of the mRNA (not shown) winds through grooves or channels on the 30S subunit surface. (b) The assembled active bacterial ribosome, viewed down into the groove separating the subunits (derived from PDB ID 2OW8, 1VSA, and 1GIX). All components are colored as in (a). (c) A pair of ribosome images in the same orientation as in (b), but with all components shown as surface renderings to emphasize the mass of the entire structure. In the structure on the right, the tRNAs have been omitted to give a better sense of the cleft where protein synthesis occurs. (d) The 50S bacterial ribosome subunit (PDB ID 1Q7Y). The subunit is again viewed from the side that attaches to the 30S subunit, but tilted down slightly compared with its orientation in (a). The active site for peptide bond formation (the peptidyl transferase activity), deep within a surface groove and far away from any protein, is marked by a bound inhibitor, puromycin (red).

2 Components required for protein-synthesis in E. coli
TABLE 27-5 Components Required for the Five Major Stages of Protein Synthesis in E. coli

3 The content of RNA and protein chains in E. coli ribosomes
TABLE 27-6 RNA and Protein Components of the E. coli Ribosome

4 Possible folding structures of 16S og 5S rRNA
FIGURE Bacterial rRNAs. Diagrams of the secondary structure of E. coli 16S and 5S rRNAs. The first (5′ end) and final (3′ end) ribonucleotide residues of the 16S rRNA are numbered.

5 The difference between bacterial and eukaryotic ribosomes
FIGURE Summary of the composition and mass of ribosomes in bacteria and eukaryotes. Ribosomal subunits are identified by their S (Svedberg unit) values, sedimentation coefficients that refer to their rate of sedimentation in a centrifuge. The S values are not necessarily additive when subunits are combined, because rates of sedimentation are affected by shape as well as mass.

6 The general structure of tRNA, the transportes of amino acids to the ribosomes
FIGURE General cloverleaf secondary structure of tRNAs. The large dots on the backbone represent nucleotide residues; the blue lines represent base pairs. Characteristic and/or invariant residues common to all tRNAs are shaded in pink. Transfer RNAs vary in length from 73 to 93 nucleotides. Extra nucleotides occur in the extra arm or in the D arm. At the end of the anticodon arm is the anticodon loop, which always contains seven unpaired nucleotides. The D arm contains two or three D (5,6-dihydrouridine) residues, depending on the tRNA. In some tRNAs, the D arm has only three hydrogen-bonded base pairs. In addition to the symbols explained in Figure 27-16: Pu, purine nucleotide; Py, pyrimidine nucleotide; G*, guanylate or 2′-O-methylguanylate.

7 3D-structure of tRNAPhe from yeast
FIGURE 27-18b Three-dimensional structure of yeast tRNAPhe deduced from x-ray diffraction analysis. The shape resembles a twisted L. (b) A space-filling model, with the same color coding (PDB ID 4TRA). The CCA sequence at the 3′ end (orange) is the attachment point for the amino acid.

8 Structure of tRNA Allows Wobble in the Third Position
FIGURE 2.16 Structure of tRNA Allows Wobble in the Third Position Transfer RNA recognizes the codons along mRNA and presents the correct amino acid for each codon. The first position of the anticodon on tRNA matches the third position of the codon. 8 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

9 three-letter system called the genetic code
The information in mRNA is translated to amino acid sequences (protein) via a three-letter system called the genetic code FIGURE 2.15 The Genetic Code The 64 codons found in mRNA are shown with their corresponding amino acids. As usual, bases are read from 5’ to 3’ so that the first base is at the 5’ end of the codon. Three codons (UAA, UAG, UGA) have no cognate amino acid but signal stop. AUG (encoding methionine) and, much less often, GUG (encoding valine) act as start codons. To locate a codon, find the first base in the vertical column on the left, the second base in the horizontal row at the top, and the third base in the vertical column on the right. 9 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

10 Transcription and translation are coupled in prokaryotes, but not in eukaryotes
FIGURE Coupling of transcription and translation in bacteria. The mRNA is translated by ribosomes while it is still being transcribed from DNA by RNA polymerase. This is possible because the mRNA in bacteria does not have to be transported from a nucleus to the cytoplasm before encountering ribosomes. In this schematic diagram the ribosomes are depicted as smaller than the RNA polymerase. In reality the ribosomes (Mr 2.7 x 106) are an order of magnitude larger than the RNA polymerase (Mr 3.9 x 105).

11 The N-terminal ends of proteins are made first
FIGURE 27-32a Polysome. (a) Four ribosomes translating a eukaryotic mRNA molecule simultaneously, moving from the 5′ end to the 3′ end and synthesizing a polypeptide from the amino terminus to the carboxyl terminus.

12 Aminoacylation of tRNA by aminoacyl-tRNA synthetase
MECHANISM FIGURE Aminoacylation of tRNA by aminoacyl-tRNA synthetases. Step 1 is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases (see Table 27-7). For class I enzymes, 2a the aminoacyl group is transferred initially to the 2′-hydroxyl group of the 3′-terminal A residue, then 3a to the 3′-hydroxyl group by a transesterification reaction. For class II enzymes, 2b the aminoacyl group is transferred directly to the 3′-hydroxyl group of the terminal adenylate.

13 General structure of amino-acyl tRNA
FIGURE General structure of aminoacyl-tRNAs. The aminoacyl group is esterified to the 3′ position of the terminal A residue. The ester linkage that both activates the amino acid and joins it to the tRNA is shaded pink.

14 The initiating amino acid at the start codon is N-formylmethionine

15 FIGURE 2.17 Translation in Prokaryotes
(A) Initiation of translation begins with the association of the small ribosome subunit with the Shine-Dalgarno sequence (S-D sequence) on the mRNA. Next, the initiator tRNA that reads AUG is charged with fMet. The charged initiator tRNA associates with the small ribosome subunit and finds the start codon. Assembly is helped by initiation factors (IF1, IF2, and IF3)—not shown. (B) During elongation peptide bonds are formed between the amino acids at the A-site and the P-site. The movement of the ribosome along the mRNA and addition of a new tRNA to the A-site are controlled by elongation factors (also not shown). (C) Termination requires release factors. The various components dissociate. The completed protein folds into its proper three-dimensional shape. 15 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

16 FIGURE 2.18 Translation in Eukaryotes
(A) Assembly of the small subunit plus initiator Met-tRNA involves the binding of factors eIF3 and eIF2. (B) The cap binding protein of eIF4 attaches to the mRNA before it joins the small subunit. (C) The mRNA binds to the small subunit via cap binding protein and the 40S initiation complex is assembled. (D) Assembly of the large subunit requires factor eIF5. After assembly, eIF2 and eIF3 depart. 16 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

17 Formation of the initiation complex in bacteria
FIGURE Formation of the initiation complex in bacteria.The complex forms in three steps (described in the text) at the expense of the hydrolysis of GTP to GDP and Pi. IF-1, IF-2, and IF-3 are initiation factors. P designates the peptidyl site, A the aminoacyl site, and E the exit site. Here the anticodon of the tRNA is oriented 3′ to 5′, left to right, as in Figure 27-8 but opposite to the orientation in Figures and

18 The Shine-Dalgarno sequence in mRNA interacts with 16 rRNA in the ribosome
FIGURE Messenger RNA sequences that serve as signals for initiation of protein synthesis in bacteria. (a) Alignment of the initiating AUG (shaded in green) at its correct location on the 30S ribosomal subunit depends in part on upstream Shine-Dalgarno sequences (pink). Portions of the mRNA transcripts of five bacterial genes are shown. Note the unusual example of the E. coli LacI protein, which initiates with a GUG (Val) codon (see Box 27-1). (b) The Shine-Dalgarno sequence of the mRNA pairs with a sequence near the 3′ end of the 16S rRNA.

19 FIGURE First elongation step in bacteria: binding of the second aminoacyl-tRNA. The second aminoacyl-tRNA (AA2) enters the A site of the ribosome bound to EF-Tu (shown here as Tu), which also contains GTP. Binding of the second aminoacyl-tRNA to the A site is accompanied by hydrolysis of the GTP to GDP and Pi and release of the EF-Tu–GDP complex from the ribosome. The bound GDP is released when the EF-Tu–GDP complex binds to EF-Ts, and EF-Ts is subsequently released when another molecule of GTP binds to EF-Tu. This recycles EF-Tu and makes it available to repeat the cycle.

20 FIGURE Second elongation step in bacteria: formation of the first peptide bond. The peptidyl transferase catalyzing this reaction is the 23S rRNA ribozyme. The N-formylmethionyl group is transferred to the amino group of the second aminoacyl-tRNA in the A site, forming a dipeptidyl-tRNA. At this stage, both tRNAs bound to the ribosome shift position in the 50S subunit to take up a hybrid binding state. The uncharged tRNA shifts so that its 3′ and 5′ ends are in the E site. Similarly, the 3′ and 5′ ends of the peptidyl tRNA shift to the P site. The anticodons remain in the A and P sites.

21 FIGURE 27-30a Third elongation step in bacteria: translocation
FIGURE 27-30a Third elongation step in bacteria: translocation. (a) The ribosome moves one codon toward the 3′ end of the mRNA, using energy provided by hydrolysis of GTP bound to EF-G (translocase). The dipeptidyl-tRNA is now entirely in the P site, leaving the A site open for the incoming (third) aminoacyl-tRNA. The uncharged tRNA dissociates from the E site, and the elongation cycle begins again.

22 FIGURE 27-31 Termination of protein synthesis in bacteria
FIGURE Termination of protein synthesis in bacteria. Termination occurs in response to a termination codon in the A site. First, a release factor, RF (RF-1 or RF-2, depending on which termination codon is present), binds to the A site. This leads to hydrolysis of the ester linkage between the nascent polypeptide and the tRNA in the P site and release of the completed polypeptide. Finally, the mRNA, deacylated tRNA, and release factor leave the ribosome, which dissociates into its 30S and 50S subunits, aided by ribosome recycling factor (RRF), IF-3, and energy provided by EF-G-mediated GTP hydrolysis. The 30S subunit complex with IF-3 is ready to begin another cycle of translation (see Figure 27-25).

23 De viktigste punktene i proteinsyntesen i bakterier:
Den første aminosyra er alltid formylmetionin, uavhengig av hva startkodonet (nesten alltid AUG eller GUG) er. Før peptidbindinger lages må aminosyrene aktiveres ved å kondensere med ATP. Denne reaksjonen drives av spalting av en fosfodiesterbinding i ATP slik at PP frigjøres. Etter dette kobles aminosyren på sitt aktuelle tRNA ved hjelp av aminoacyl tRNA syntetase. 30S subenheten av ribosomet bindes til mRNA slik at AUG blir posisjonert i P-setet (peptidylsetet). Husk at det er her Shine-Dalgarno sekvensen i mRNA kommer inn ved å basepare med en del av 16S rRNA. tRNA med formylmetionin rekrutteres til P-setet ved binding til AUG i mRNA. 50S subenheten rekrutteres til komplekset. Neste aminosyre rekrutteres via sitt tRNA til kodon 2 i mRNA ved A-setet (aminoacyl-setet) i ribosomet. En peptidbinding dannes ved at formylmetionin linkes til aminosyren i A-setet. tRNA med dipeptidet translokeres fra A-setet til P-setet. En tredje aminosyre rekrutteres via sitt tRNA til kodon 3 i mRNA ved A-setet. Ny peptidpinding dannes som over. Prosessen fortsetter til stopp-kodonet. Dette senses av release faktor og proteinet forlater ribosomet i Exit site. Ribosomet spaltes i 30S og 50S subenheter igjen. Det inngår mange proteinfaktorer (initieringsfaktorer, elongeringsfaktorer etc) i de ulike stegene, men dere trenger ikke å huske navnene på disse eller hvor de inngår. Derimot er det svært viktig å være klar over at det forbrukes veldig mye GTP. Siden det lages veldig mye proteiner i levende celler blir dermed proteinsyntesen en energimessig sett veldig kostbar prosess.

24 Proteins and folding and posttranslatoric modification
Proteins can be denatured, i.e by heating. This means that the folding structure collapses, resulting in loss of functionality Proteins from thermophilic organisms can resist boiling Some proteins fold correctly by themselves after retransfer to low temperatures, but most proteins don’t During production many proteins can only obtain correct folding if assisted by other proteins (chaperones) during production in living cells If large quantities of a specific chaperone-dependent protein is produced in a cell, it may become misfolded. This represents a very serious problem in biotechnology If a protein is misfolded during production it may in some, but not all cases, be correctly refolded in the laboratory Many proteins, particularly from eukaryotes, are further modified in the cells after translation. This can involve phosphorylation, glycosylation etc. The modifications have various biolological functions, and this is also very important in biotechnology.