Translation: Protein Synthesis,Protein synthesis machinery: mRNA tRNA Aminoacyl-tRNA synthetases Ribosome Genetic code: Cracking the genetic code Codon usage Translation reaction: Initiation Elongation Termination Difference between eukaryotes and prokaryotes in translation Protein sorting Inhibition of protein synthesis by antibiotics,Outline,Peptide bond formation,Translation: biosynthesis of proteins,Translation is among the most conserved mechanisms across all organisms It is the most energetically costly for the cell. Protein synthesis consumes about 80% energy of the cell 50,000 ribosomes per E. coli cell Ribosomes are the most complex polymerases. Molecular weight of a ribosome in E. coli is 2.7 million Daltons Two thirds of the ribosome is RNA; Ribosomes can been see under EM Translation requires: amino acids, mRNAs, tRNAs, aminoacyl tRNA synthetases, ribosomes,Protein synthesis machinery,The three roles of RNA in protein synthesis. Messenger RNA (mRNA) is translated into protein by the joint action of transfer RNA (tRNA) and the ribosome, which is composed of numerous proteins and two major ribosomal RNA (rRNA) molecules.,Three roles of RNA in translation,Messenger RNA (mRNA) carries the genetic information copied from DNA in the form of a series of three-base code “words,“ each of which specifies a particular amino acid. Transfer RNA (tRNA) is the key to deciphering the code words in mRNA. Each type of amino acid has its own type of tRNA, which binds it and carries it to the growing end of a polypeptide chain if the next code word on mRNA calls for it. The correct tRNA with its attached amino acid is selected at each step because each specific tRNA molecule contains a three-base sequence that can base-pair with its complementary code word in the mRNA. Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes. These complex structures, which physically move along an mRNA molecule, catalyze the assembly of amino acids into protein chains. They also bind tRNAs and various accessory molecules necessary for protein synthesis. Ribosomes are composed of a large and small subunit, each of which contains its own rRNA molecule or molecules.,mRNA,ORF (open reading frame): The protein coding region(s) of each mRNA is composed of a continous non-overlapping string of codons called an open reading frame (ORF). Each ORF specifies a single protein and starts with a start codon and ends with a stop codon. In bacteria, start codons include 5-AUG-3 (the most common), 5-GUG-3 and 5-UUG-3. In eukaryotes, the start codon is always 5-AUG-3. The start codon not only specifies the first amino acids (aa) to be added, but it also defines the reading frame for subsequent codons. Eukaryotic mRNAs mostly contain a single ORF (monocistronic mRNA), Prokaryotic mRNA usually contain multiple ORF (polycistronic mRNAs). Prokaryotic mRNAs have a ribosome binding site (RBS) also called Shine-Dalgarno sequence. This sequence is complementary to the 16S rRNA of the small ribosomal subunit. Eukaryotic mRNAs have a 5 cap that binds ribosomes with the help of initiator proteins. Once bound, the ribosome scans the mRNA until it finds the first 5-AUG-3. Translation in eukaryotes is enhaced by the Kozak sequence: GCCACCAUGG and by the presence of a poly A tail.,mRNA,tRNA,tRNAs are about 75-95 nucleotides long. The sequence from tRNA to tRNA varies, but there are common features to all tRNAs: a 3 terminus 5-CCA-3 (where the aa attaches), the presence of unusual bases and a common secondary structure. The secondary structure resembles a cloverleaf where there is an acceptor stem (5-CCA-3), three stem loops and the anticodon loop. Attachment of aa to tRNA. tRNA molecules are charged when they have an aa attached. Adenylylation: an amino acid reacts with ATP and becomes adenylylated (AMP-aa). A pyrophaosphate is released. This reaction is catalyzed by the aa specific tRNA synthetase. tRNA charging: The adenylylated aa which remains tightly bound to the synthetase reacts with the tRNA. The energy released when the bond is broken helps drive the formation of the peptide bond that links aa to each other.,Structure of tRNAs. (a) The primary structure of yeast alanine tRNA (tRNAAla), the first such sequence determined. This molecule is synthesized from the nucleotides A, C, G, and U, but some of the nucleotides, shown in red, are modified after synthesis: D = dihydrouridine, I = inosine, T = thymine, Y = pseudouridine, and m = methyl group. Although the exact sequence varies among tRNAs, they all fold into four base-paired stems and three loops. The partially unfolded molecule is commonly depicted as a cloverleaf. Dihydrouridine is nearly always present in the D loop; likewise, thymidylate, pseudouridylate, cytidylate, and guanylate are almost always present in the TYCG loop. The triplet at the tip of the anticodon loop base-pairs with the corresponding codon in mRNA. Attachment of an amino acid to the acceptor arm yiel