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 mRNARBS: 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).

(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 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.

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

U D

tRNAs share a common secondary structure that resembles a cloverleaf

Fig 14-4 cloverleaf representation of the 2nd structure of tRNA

(1) The acceptor stem

U loop: 5’-TUCG-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 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

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 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

Fig 14-11 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 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.

Fig 14-13 Composition of the prokaryotic and eukaryotic ribosomes.

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 14-18A: for aminoacylated-tRNAP: for peptidyl-tRNAE: 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.

Fig 14-21 The polypeptide exit center

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

Fig 14-24 methionine and N-formyl methionine

• Initiator tRNA: fMet-tRNAifMet (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 14-25 A summary of translation initiation in prokaryotes

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

The start codon is found by scanning downstream from the 5’ end of the mRNA

Fig 14-27 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 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.

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 14-35Left: EF-Tu-GDPNP-Phe-tRNARight: 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 translationrelease 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 codonClass 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 GTPprokaryotes: RF3eukaryotes: eRF3

Short regions of class I release factors recognize stop codons and trigger release of the peptidyl chain

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 14-39 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 14-40 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.

Regulation of translation

• Although the expression of most genes is regulated at the level of mRNA transcription, it is more effective for the cell to regulate gene expression at the level of translation.

• As with other types of regulation, translational control typically functions at the level of initiation.

Protein or RNA binding near the ribosome-binding site negatively regulates bacterial translation initiation

Regulation of prokayryotic translation: ribosomal proteins are translational repressors of their own synthesis

Global regulators of eukaryotic translation target key factors required for mRNA recognition and initiator tRNA ribosome binding

Spatial control of translation by mRNA-specific 4E-BPs

An iron-regulated, RNA-binding protein controls translation of ferritin

Translation of the yeast transcriptional activator Gcn4 is controlled by short upstram ORFs and ternary complex abundance

Fig 14-48

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 tRNAala.

The SsrA RNA rescues ribosomes that translate broken mRNAs

Fig 14-39 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