EFB325 Cell Physiology

Ribosomes, tRNAs, and translation

In both prokaryotes and eukaryotes, protein synthesis is accomplished by ribosomes, which bind to mRNA, using it as a template, and also binding to tRNAs, which are "adaptors" between the codons and the proper amino acids. Translation is very similar in prokaryotes and eukaryotes; there are only a few minor differences.

Ribosome assembly

Ribosomes are complexes of RNA and protein, and are composed of a large subunit and a small subunit.

In both eukaryotes and prokaryotes, there are rRNAs transcribed as one RNA, which must then be cleaved

• prokaryotic ribosomes have 3 rRNAs; eukaryotic ribosomes have 4 rRNAs
• 3 rRNAs are transcribed as one RNA transcript, the three rRNAs are released when the RNA transcript is cleaved (the regions between the rRNAs are called spacer regions)
• in eukaryotes: the genes for the three large rRNAs are transcribed by RNA polymerase I and the smallest tRNA gene by RNA polymerase III (but not RNA polymerase II, which is used for mRNAs)

The rRNAs assemble together with many different small ribosomal proteins to form the large and small subunits

• eukaryotic ribosomes are slightly more complex and a little larger than prokaryotic ribosomes

tRNA processing and structure

The tRNAs represent the adaptors linking the codons in mRNA to the proper amino acid for protein synthesis, which occurs on ribosomes.

• the tRNAs are transcribed, then undergo some processing and folding to form their final functional 3D structure
• there are four regions of the tRNA which are base-paired (and are double-stranded) to form stem-loops; these loops contain some chemically modified bases and are important for distinguishing the different tRNAs
• one of these loops contains the anticodon, the 3-base sequence that is the complement of the codon sequence in the mRNA

The proper amino acid is attached to the correct tRNA by the aminoacyl-tRNA synthetase

• there are only 20 different aminoacyl-tRNA synthetases=1 specific for each amino acid, but each one can recognize all of the tRNAs which are used for each amino acid
• this protein has binding sites specific for the amino acid and specific for the anticodon(s) of the tRNA(s)
• it uses energy from ATP to attach the amino acid to the 3' end of the tRNA (thus the amino acid is activated)

The tRNA anticodon base-pairs with the codon of an mRNA, but there is "wobble"

• the three base sequence in the anticodon is complementary to the codon sequence in mRNA; we will express the anticodon sequence by reading 3'->5', so that the bases match up with the codon sequence, which we read 5'->3'
• the base-pairing of the tRNA anticodon to the mRNA codon is not as strict as the base-pairing of double-stranded DNA; so one tRNA can recognize more than one codon
• this means that even though there are 61 codons for amino acids, there are far less than 61 (as few as 31) different tRNAs
• the flexibility in base-pairing of anti-codon to codon is at the 3^rd base (3' side) of the codon, which base-pairs with the 5'-base of the anticodon

The wobble rules

The ribosome has 3 sites where tRNA can bind and a site that binds the mRNA

• the A (aminoacyl) site is where an incoming aminoacyl-tRNA binds and base-pairs with a codon of the mRNA - the specificity of binding of the aminoacyl tRNAs is conferred by the codon-anticodon base-pairing (not by the actual amino acid linked to the tRNA)
• the P (peptidyl) site is where the aminoacyl-tRNA holding the growing polypeptide chain is bound; the tRNA is base-paired with the mRNA codon next to the codon in the A site
• the E (exit) site is where the tRNA (now without an amino acid) leaves the ribosome complex

Translation starts at a start codon

• translation does not start at the first base of the 5' end of an mRNA, there is a region of the transcript which is not translated=leader; translation starts at a start codon, which is almost always AUG (encodes methionine)
• methionine (a modified methionine in bacteria) is the first amino acid in nearly all proteins

Prokaryotes and eukaryotes use a slightly different mechanism to identify the start codon

In prokaryotes

• there is a region of the 16S rRNA (in the 30S ribosomal subunit) that can base pair with a special sequence in the mRNA, just before the start codon. This sequence is called the Shine-Dalgarno sequence, and it positions the ribosome on the mRNA to begin at the start codon

In eukaryotes

• the ribosome small subunit binds to the 5' cap, then scans toward the 3' end until it finds a start codon (moves to the first AUG)

Initiation of translation is a step-wise process

• initiation (in both prokaryotes and eukaryotes) involves the binding of the small ribosomal subunit with other proteins called initiation factors (IFs), which assist the ribosomal subunit to bind to the mRNA

in prokaryotes

• the ribosomal subunit binds to the mRNA first, then the initiator tRNA binds at the start codon
• the initiator tRNA carries a modified methionine

in eukaryotes

• the initiator tRNA binds with the small ribosomal subunit and the IFs, then this complex scans to the first start codon
• the initiator tRNA carries methionine

in both systems

• when the initiator tRNA is bound to the start codon, then the large ribosomal subunit binds, and the IFs are released
• the initiator tRNA is bound to the P site of the ribosome
• energy from GTP, which binds to one of the IFs, helps drive the initiation process forward

Elongation of the polypeptide occurs by the addition of an amino acid at the A site to the polypeptide chain bound in the P site

binding of aminoacyl tRNA

• the appropriate aminoacyl tRNA will fill the open A site of the ribosome, with the anticodon base-pairing to the next codon
• the aminoacyl tRNA is carried there by a protein called elongation factor (EF), which is released after the aminoacyl tRNA binds to the A site
• positioning of the aminoacyl tRNA in the A site is driven forward by energy from 2 GTPs bound to the EF protein (the GTPs are hydrolyzed to GDPs)

peptide bond formation

• the peptide bond is formed between the carboxyl group of the amino acid bound to the tRNA in the P site (this amino acid is at the end of the growing polypeptide chain) and the amino group of the amino acid bound to the tRNA in the A site
• the bond between the amino acid and tRNA in the P site is broken in this reaction, and the energy from that bond drives the reaction forward
• this reaction is catalyzed by one of the rRNA molecules of the ribosomal large subunit (an example of a ribozyme)

translocation of the ribosome

• a different EF protein helps the tRNAs shift sites within the ribosome using energy from the hydrolysis of GTP; the P site tRNA moves to the E site (then is released); the A site tRNA (which is carrying the polypeptide) shifts to the P site
• since the tRNA is base-paired to the mRNA, then the mRNA is pulled three bases forward (toward the 3' end) to position the next codon in the A site
• now the A site is open again and ready for the next aminoacyl tRNA

Translation ends at a stop codon

• translation will proceed through the coding sequence of the gene until coming to a stop codon (UGA, UAA, or UAG) for which there are no tRNAs
• (the portion of the transcript beyond the stop codon is the trailer sequence)
• instead of a tRNA, when a stop codon is positioned in the A site of the ribosome, a protein called the release factor binds in the A site
• when release factor binds, the bond between the carboxyl-end of the polypeptide and the tRNA in the P site is hydrolyzed, releasing the completed polypeptide
• the ribosome subunits then dissociate from the mRNA

Prokaryotes vs. eukaryotes

• Note that since prokaryotic ribsomes bind directly to the mRNA immediately before the start codon, then multiple genes can be transcribed together on one mRNA, and all the genes will be translated simultaneously (this arrangement of genes together, all with one promoter, is called an operon)
• Eukaryotic ribsomes bind to the 5' cap and scan down the mRNA to the first start codon, thus only one gene can be contained on an mRNA transcript (if there was a second gene downstream of the first, it would never be found by a ribosome) - thus, there are no operons in eukaryotes

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