Ch14 Translation • Messenger RNA • Transfer RNA • Attachment of amino acids to tRNA • The ribosome • Initiation of translation • Translation elongation • Termination of translation • Regulation of translation • Translation-dependant regulation of mRNA and protein stability
Messenger RNAPolypeptide chains are specified by open-reading frames Fig 14-1 Three possible reading frames of the E. coli trp leader sequence
Open reading frame (ORF): a contiguous string of codons that specify a single protein; read in a particular frame (as set by the first codon) that is open to translation. ORF starts and ends at internal sites within the mRNA. • Start codon in eukaryotes: AUG • Stop codons: UAG, UGA, UAA • Eukaryotic mRNAs almost always contain a single ORF, whereas prokaryotic mRNAs contain one or more ORF.
Polycistronic mRNA: mRNA that contain multiple ORF. • Monocistronic mRNA: mRNA that contain single ORF.
Fig 14-2 Structure of mRNA RBS: ribosome binding site
Prokaryotic mRNAs have a ribosome binding site that recruits the translational machinery • Ribosome binding site = Shine-Dalgarno sequence 1. 3-9 bp on the 5’ side of the start codon 2. Complementary to a sequence near the 3’ end of 16s rRNA.
Eukaryotic mRNAs are modified at their 5’ and 3’ ends to facilitate translation • 5’ modifications: (1) Eukaryotic mRNAs recruit ribosomes using 5’ cap. 5’ cap: methylated G nucleotide that is linked to 5’ end of mRNA by 5’-5’ linkage • 5’ cap recruits ribosome to the mRNA; the ribosome bound to mRNA moves in a 5’ to 3’ direction until it encounters a start codon (scanning). • Kozak sequence (5’-G/ANNAUGG-3’): thought to interact to with initiator tRNA
3’ modifications: Poly-A tail enzymatically added by poly-A polymerase. enhance translation by promoting efficient recycling of ribosomes.
Transfer RNAtRNAs are adaptors between codons and amino acids • tRNA: between 75 to 95 ribonucleotides • tRNA end at 3’-terminus with the sequence CCA, where the cognate amino acid is attached. • Unusual bases are present in tRNA structure.
yU D Fig 14-3 A subset of modified nucleosides found in tRNA
tRNAs share a common secondary structure that resembles a cloverleaf (1) The acceptor stem (2) yU loop: 5’-TyUCG-3’ (3) D loop (4) anticodon loop (5) variable loop: 3-21 bases Fig 14-4 cloverleaf representation of the 2nd structure of tRNA
tRNAs have an L-shaped 3-D structure Fig 14-5 Conversion between the cloverleaf and the actual 3-D structure of a tRNA
Attachment of amino acids to tRNAtRNAs are charged by the attachment of an amino acid to the 3’ terminal adenosine nucleotide via a high-energy acyl linkage • Charged tRNA • Uncharged tRNA
Aminoacyl tRNA synthetase charge tRNAs in two steps Adenylylation of amino acid Fig 14-6
Each aminoacyl tRNA synthetase attaches a single amino acid to one or more tRNAs isoaccepting tRNA • tRNA synthetase recognize unique structural features of cognate tRNAs Fig 14-7
Fig 14-8 co-crystal structure of glutaminyl aminoacyl tRNA synthetase with tRNAgln
Aminoacyl-tRNA formation is very accurate Fig 14-9 Some aminoacyl tRNA synthetases use an editing (as a molecular sieve) pocket to charge tRNAs with high fidelity
The ribosome is unable to discriminate between correctly and incorrectly charged tRNAs Fig 14-10 cysteinyl-tRNA charged with C or A
The Ribosome • Rate of DNA replication: 200-1000 nt/sec • Rate of translation in prokaryotes: 2-20 amino acids/sec • Rate of translation in eukaryotes: 2-4 amino acids/sec
In prokaryotes, the transcription and translation machineries are located in the same compartment. Fig 14-11 prokaryotic RNA polymerase and the ribosome at work on the same RNA
In eukaryotes, transcription happens in the nucleus while translation happens in the cytoplasm.
The ribosome is composed of a large and a small subunit Fig 14-12 sedimentation by ultracentrifugation to separate individual ribosome subunits and the full ribosomes. S: Svedberg (sedimentation velocity) determined by both size and shape.
Large subunit contains peptidyl transferase center (for formation of peptide bond) • Small subunit contains decoding center.
The large and small subunits undergo association and dissociation during each cycle of translation Fig 14-14 Overview of the events of translation
Fig 14-15 An mRNA bearing multiple ribosomes is known as a polyribosome or a polysome.
New amino acids are attached to the C-terminus of the growing polypeptide chain Peptides bonds are formed by transfer of the growing polypeptide chain from one tRNA to another
Fig 14-16 The peptidyl transferase reaction The ribosome catalyzes a single chemical reaction: The formation of a peptide bond
Fig 14-17 two views of the ribosomes • Ribosomal RNAs are both structural and catalytic determinants of the ribosome. • Most ribosomal proteins are on the periphery of the ribosome, while the functional core of ribosome is composed mostly from rRNA.
The ribosome had three binding sites for tRNA Fig 14-18 A: for aminoacylated-tRNA P: for peptidyl-tRNA E: for exit
Channels through the ribosome allow the mRNA and growing polypeptide to enter and/or exit the ribosome (Fig 14-19) Fig 14-20 The interaction between the A site and P site tRNAs and the mRNA within the ribosome.
The initiation of translation Fig 14-22 An overview of the events of translation initiation
Prokaryotic mRNAs are initially recruited to the small subunit by base-pairing to rRNA Fig 14-23 The 16S rRNA interacts with the RBS to position the AUG in the P site.
A specialized tRNA charged with a modified methionine binds directly to the prokaryotic small subunit • Initiator tRNA: fMet-tRNAifMet (base-pairs with AUG or GUG) • Deformylase removes the formal group during or after the synthesis Fig 14-24 methionine and N-formyl methionine
Three initiation factors direct the assembly of an initiation complex that contains mRNAs and the initiator tRNA A model of initiation factor binding to the 30S ribosomal subunit. IF1: prevents tRNA from binding to A site IF2: a GTPase; interacts with initiator tRNA and IF1, and thus prevents further tRNA binding to small subunits. IF3: binds to small subunit and prevent it from reassociating with large subunit; essential for translation initiation.
Eukaryotic ribosomes are recruited to the mRNA by the 5’ Cap Fig 14-27 assembly of the eukaryotic small ribosomal subunit and initiator tRNA onto the mRNA eIF4B: helicase; unwinding any RNA secondary structure
Fig 14-27 identification of the initiating AUG by the eukaryotic small ribosomal subunits The start codon is found by scanning downstream from the 5’ end of the mRNA
uORF: short, upstream, open-reading frame, less than 10 amino acids long
Translation initiation factors hold eukaryotic mRNAs in circles Fig 14-28 a model for the circularization of eukaryotic mRNA, through the interaction between eIF4G and poly-A binding protein.
Translation elongation Fig 14-29 summary of the steps of translation The mechanism of elongation is highly conserved between prokaryotes and eukaryotes.
Aminoacyl-tRNA are delivered to the A site by elongation factor EF-Tu. Fig 14-30 EF-Tu escorts aminoacyl-tRNA to the A site of the ribosome. The interaction between EF-Tu and factor binding site of large subunit triggers the GTPase of EF-Tu.
The ribosome uses multiple mechanisms to select against incorrect aminoacyl-tRNAs.
The ribosome is a ribozyme: peptidyl transferase reaction is catalyzed by RNA, mainly 23S rRNA.
Peptide bond formation and the elongation factor EF-G drive translocation of the tRNAs and the mRNA