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 RNA Polypeptide 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 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). (2)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 RNA tRNAs 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.

Fig 14-3 A subset of modified nucleosides found in tRNA UU D

tRNAs share a common secondary structure that resembles a cloverleaf Fig 14-4 cloverleaf representation of the 2 nd structure of tRNA (1) The acceptor stem  U loop: 5’-T  UCG-3’ (3) D loop (4) anticodon loop (5) variable loop: 3-21 bases

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 tRNA tRNAs 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 Fig 14-6 Adenylylation of amino acid

Transfer of adenylylated amino acid to tRNA: tRNA charging

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 tRNA gln

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 cysteinyl-tRNA charged with C or A

The Ribosome Rate of DNA replication: nt/sec Rate of translation in prokaryotes: 2-20 amino acids/sec Rate of translation in eukaryotes: 2-4 amino acids/sec

Fig prokaryotic RNA polymerase and the ribosome at work on the same RNA In prokaryotes, the transcription and translation machineries are located in the same compartment.

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 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.

Fig Composition of the prokaryotic and eukaryotic ribosomes.

The large and small subunits undergo association and dissociation during each cycle of translation Fig Overview of the events of translation

Fig 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 The peptidyl transferase reaction The ribosome catalyzes a single chemical reaction: The formation of a peptide bond

Fig 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 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 The interaction between the A site and P site tRNAs and the mRNA within the ribosome.

Fig The polypeptide exit center

The initiation of translation Fig An overview of the events of translation initiation

Prokaryotic mRNAs are initially recruited to the small subunit by base-pairing to rRNA Fig 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 Fig methionine and N-formyl methionine Initiator tRNA: fMet-tRNA i fMet (base-pairs with AUG or GUG) Deformylase removes the formal group during or after the synthesis

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.

Fig A summary of translation initiation in prokaryotes

Eukaryotic ribosomes are recruited to the mRNA by the 5’ Cap Fig assembly of the eukaryotic small ribosomal subunit and initiator tRNA onto the mRNA eIF4B: helicase; unwinding any RNA secondary structure

The start codon is found by scanning downstream from the 5’ end of the mRNA Fig identification of the initiating AUG by the eukaryotic small ribosomal subunits

uORF: short, upstream, open-reading frame, less than 10 amino acids long

IRES (internal ribosome entry site)

Translation initiation factors hold eukaryotic mRNAs in circles Fig a model for the circularization of eukaryotic mRNA, through the interaction between eIF4G and poly-A binding protein.

Translation elongation Fig 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 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.

Minor groove interactions

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

Hybrid state of tRNA exposes factor-binding site; EF-G can only bind to ribosome by a GTP-bound form. EF-G drives translocation by displacing the tRNA bound to the A site

Fig Left: EF-Tu-GDPNP-Phe-tRNA Right: EF-G-GDP How does EF-G-GDP interact with the A site so effectively?

EF-Tu-GDP and EF-G-GDP must exchange GDP for GTP prior to participating in a new round of elongation A cycle of peptide bond formation consumes two molecules of GTP and one molecule of ATP GDP has a lower affinity for EF-G than GTP For EF-Tu, a GTP-exchange factor EF-Ts is required for the GDP-GTP exchange. Fig14-6

Termination of translation release factors terminate translation in response to stop codons Release factors (RF) activates the hydrolysis of polypeptide from the peptidyl-tRNA Class I RF: recognizes stop codon Class II RF: stimulate dissociation of class I RF from ribosome Class I RF: prokaryotes: RF1 (UAG, UAA); RF2 (UGA, UAA) eukaryotes: eRF1 (UAG; UGA; UAA) Class II RF: regulated by GTP prokaryotes: RF3 eukaryotes: eRF3

Short regions of class I release factors recognize stop codons and trigger release of the peptidyl chain Fig Model of a RF1 bound to the A site GGQ: involved in polypeptide hydrolysis; close to peptidyl- transferase center SPF: peptide anticodon; for interacting with stop codon

GDP-GTP exchange and GTP hydrolysis control the function of the class II release factor Fig polypeptide release is mediated by two RF RF-3 has a higher affinity to GDP than to GTP

The ribosome recycling factor (RRF) mimics a tRNA Fig RRF and EF-G combine to stimulate the release of tRNA and mRNA from a terminated ribosome. RRF is only associated with the large subunit of the A site.

Puromycin terminates translation by mimicing a tRNA in the A site.

Translation-dependent regulation of mRNA and protein stability Being single-stranded, mRNAs are more susceptible to breakage. Such damaged mRNAs have the possibility of making incomplete or incorrect proteins.

tmRNA: in prokaryotic cell, stalled ribosomes are rescued by a chimeric RNA (part tRNA and part mRNA) SsrA is a 457 nt tmRNA that includes a 3’ end strongly resembles tRNA ala. The SsrA RNA rescues ribosomes that translate broken mRNAs

Fig The tmRNA and SsrA rescue ribosomes stalled on prematurely terminated mRNAs. How does the SsrA RNA bind to only stalled ribosomes?? Large size

Eukaryotic cells degrade mRNAs that are incomplete or that have premature stop codon Fig 14-40

Exosome: 3’-5’ exonuclease