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Department of Biological and Environmental Sciences

Cell & Molecular Biology
Dr. David A. Johnson
Biol 405    4 Credits   Spring 2017  MWF 11:45-12:50 AM   PH

Protein Synthesis

"...alanine tRNA appears to recognize GCU, GCC and GCA ... I have suggested that this is because of a "wobble" in the pairing in the third place..."
Francis H. C. Crick (1966)
The genetic information in the form of a sequence of bases in DNA is transcribed into individual "messages" in the form of a sequence of bases in RNA. This process of transcription is followed by the process of translation, in which the meaning of this informational RNA molecule is decoded. Translation is then the process by which an RNA base sequence is used to directed the synthesis of a specific polypeptide with a specific amino acid sequence. In eukaryotes, it follows RNA processing and occurs after the mRNA leaves the nucleus through a nuclear pore and travels to the cytoplasm. That is, the two processes, transcription and translation, are temporally and spatially separated. In prokaryotes, however, translation begins before transcription is finished (they occur simultaneously) and they occur in the same place. In order for translation to occur, several things must be present--the major ones being an mRNA molecule, a ribosome, and charged transfer RNAs (tRNAs).
  • Transfer RNAs: These single-stranded RNAs are 70-80 bases in length and all have common (conserved) sequences. There are numerous tRNAs--at least one for every amino acid. Eukaryotic and prokaryotic tRNAs are very similar in many respects. tRNAs have many modified bases, as we learned in the last unit. An amino acid will become covalently bonded to its specific tRNA creating a charged tRNA. Each tRNA is designated with a superscript that indicates which amino acid it will bind. For example, tRNAala is the tRNA that will be charged with (bound to) alanine. When it is charged, it is indicated as ala-tRNAala (alanine attached to the alanine-specific tRNA).
    • tRNA's 3-D Structure: tRNAs all have the same general shape, described as a cloverleaf with internal base pairing holding the cloverleaf in place. This cloverleaf actually folds in on itself producing a more complex 3-D structure.
    • 3' CCA: All tRNAs have the trinucleotide CCA at the 3' terminus (as we saw in the last topic). It is here that the amino acid will attach. The 3' CCA is at one end of the folded tRNA and the anticodon (see below) is at the other end.
    • Aminoacyl-tRNA Synthetases: The attachment of an amino acid to the tRNA is catalyzed by a group of enzymes called aminoacyl-tRNA synthetases. There is a specific enzyme for each amino acid/tRNA pair. Aminoacyl-tRNA synthetase is the generic name, with each amino acid having a specific enzyme. For example, the enzyme that charges tRNAala is alanyl-tRNA synthetase. A specific tRNA synthetase is capable of binding specifically to both the designated tRNA and to the designated amino acid. This reaction requires ATP and occurs in two steps. First, tRNA synthetase activates the amino acid by adding an AMP onto the carboxyl end of the amino acid (using ATP and releasing pyrophosphate). The carboxyl end is attached to the remaining phosphate of the AMP. The amino acid is then transferred to the oxygen at the 2' or 3' carbon of the 3' end of the tRNA (the A nucleotide of the CCA). (Type I and type II synthetases, respectively.) Even if the amino acid is initially attached to the 2' OH, it is the 3' OH form that is used in protein synthesis.* This charged tRNA is now ready to take part in translation. There is one aminoacyl-tRNA synthetase for each amino acid (20 of them) in most organisms. The one enzyme recognizes more than one tRNA if there are cognate tRNA for an amino acid. In bacteria, however, there may be less than 20 aminoacyl-tRNA synthetases**!

    • Anticodon: Three bases in the middle of the tRNA sequence make up the anticodon (although they end up at one end of the tRNA after it forms the cloverleaf and folds up). These three bases are unique for each tRNA and (as we will see) hydrogen bond to the codon of the mRNA during translation. (See Degenerate below for the role of modified bases in the anticodon.)
  • The Ribosome: Ribosomes are the sites where the actual protein synthesis occurs and are similar in composition in prokaryotes and eukaryotes. Like all large molecules and particles, the size of ribosomes can be designated as a sedimentation velocity. Prokaryotic ribosomes are 70S particles and eukaryotic ribosomes are 80S particles. Ribosome are the smallest and the most abundant of the organelles (20,000 in E. coli and as many as 10 x 106 in some eukaryotic cells). Ribosomes are composed of rRNA and ribosomal proteins. The rRNAs have a 3-D structure even more complex than that of the tRNAs (rRNAs are much larger than tRNAs).
      • Prokaryotic Ribosomes: E. coli's ribosomes are composed of a 50S and a 30S subunit. The RNAs in the larger subunit are the 23S and 5S rRNAs (see last unit) and it has 34 ribosomal proteins. The smaller subunit has the 16S rRNA and 21 ribosomal proteins.
      • Eukaryotic Ribosomes: The subunits of a eukaryotic ribosome are slightly larger than E. coli's: the larger subunit is 60S and the smaller one is 40S. The 60S subunit includes the 28S, the 5.8S, and the 5S rRNA plus about 45 ribosomal proteins. The 40S subunit has the 18S rRNA and about 30 ribosomal proteins.
    • Ribosome Function: As we will see below, the rRNAs and ribosomal proteins are important in various binding and catalytic activities of translation.
  • mRNA: The mRNA molecule used in translation has several features. (Remember, in eukaryotes it has already undergone extensive processing, including splicing.)
    • Untranslated Regions: Even after processing , mRNA molecules include a region before the beginning of the coding sequence and after the coding sequence ends. The "front" end of the mRNA is referred to as the 5' untranslated region (5' UTR) and the sequence at the "back" end is called the 3' untranslated region (3' UTR).
    • Polycistronic mRNAs: Prokaryotes often have long mRNAs that code for more than one polypeptide. These are referred to as polycistronic mRNAs. Ribosome may dissociate from the mRNA at the end of translation of a polypeptide, or stay on the mRNA and continue translating the next CDS. (About 73% of E. coli's promoters control a coding unit, meaning the rest control polycistronic mRNA transcription.)(Some rare cases of something similar occur in eukaryotes, but do not use the same mechanism.)
  • Translation: The process of translation is divided into these three stages:
    • Initiation: During initiation in both prokaryotes and eukaryotes, a special charged tRNA and the 5' end of the mRNA bind to the small ribosomal subunit. Initiation in both prokaryotes and eukaryotes involves various protein factors.
      • Prokaryotes: Initiation in prokaryotes begins with the binding of 3 initiation factors to the small subunit (IF2 has GTP bound to it). Then, the mRNA and the special charged tRNA bind to the complex. IF2 recognizes this special tRNA. This tRNA is the initiator tRNA called tRNAfmet. It is charged with a special amino acid: N-formyl methionine (fmet-tRNAfmet). This methionine has been modified by having a formyl group (H-C=O) added to its N-terminus, making it impossible to join with the C-terminus of a previous polypeptide (only needed in prokaryotes. Why?). A sequence (the Shine-Dalgarno sequence: AGGAGG in most prokaryotes, AGGAGGU in E. coli) on the mRNA is recognized by a 16S rRNA sequence near its 3' end. The ribosomal subunit then scans downstream from this sequence on the mRNA until it finds the AUG initiator codon (5-9 nucleotides downstream), where translation will actually begin. Since prokaryotes can have more than one gene per mRNA, this means that the small ribosomal subunit will also be able to recognize an internal Shine-Dalgarno sequence and begin to make a second (or third) polypeptide. The 50S ribosomal subunit then joins and the IF2 is released and its bound GTP is hydrolyzed to GDP + P and initiation is complete.


      • Eukaryotes: During initiation in eukaryotes, the tRNA and small ribosomal subunit binds to the 5' 7-methylguanosine cap (instead of the Shine-Dalgarno sequence) and scans from there until it encounters the AUG initiation codon. There are numerous protein factors required for events like the binding to the small ribosomal subunit, binding to the charged initiator tRNA (in eukaryotes: met-tRNAmet), recognition of the 5' cap, and recognition of the 3' poly-A tail (eukaryotic initiation involves both the 5' and 3' end of the mRNA and its folding). After finding the AUG codon, the 60S subunits joins, GTP is hydrolyzed and initiation is complete.

    • Elongation: In both eukaryotes and prokaryotes, the assembled ribosome has three potential tRNA binding sites: the A site (aminoacyl site), the P site (peptidyl site) and the E site (exit site). The charged initiator tRNA (fmet-tRNAfmet or met-tRNAmet) is bound to the P site (which is between the other two sites). Then, the next charged tRNA binds to the A site. This binding is directed by the codon-anticodon base pairing (antiparallelly aligned). An RNA of the small ribosomal subunit checks to make sure this pairing is correct. Then, a GTP which was bound to an elongation factor (there are many of them) is hydrolyzed and that factor is released. Next, a peptide bond is formed between the two adjacent amino acids, with the amino acid in the P site (its C-terminus is bound to the 3' end of its tRNA) being transferred to the N-terminus of the adjacent amino acid. The tRNA at the P site has been released from its amino acid and the one at the A site now has 2 amino acids attached to its 3' end. This catalytic activity is actually carried out by RNA that is part of the 50S subunit. Next, translocation occurs in which the ribosome "moves" down the mRNA so that the tRNA that was in the P site is now in the E site (it exits) and the one that was in the A site is now in the P site. This also requires GTP hydrolysis and elongation factors. With the A site now open, this process can be repeated and a polypeptide made (15 amino acids/second). Several ribosomes (a polysome or polyribosome) may be translating a given mRNA at any time.

    • Termination: When a codon for which there is no tRNA with a complementary anticodon comes into the A site, translation terminates. These terminator codons are UAA, UAG, and UGA (also called nonsense codons). Instead of a tRNA binding to the open A site, a release factor binds there and stops protein synthesis. In prokaryotes there are two release factors that do this work (one recognizes UAA and UAG, the other recognizes UAA or UGA). In eukaryotes, one factor recognizes all three terminator codons. (A term you might run across: ORF = open reading frame = a long DNA sequence that could be transcribed into functional mRNA = a potential "structural gene." That is, it has an initiator codon and no terminator codon in the same reading frame for some distance. Therefore  it could potentially be a polypeptide-coding region.)
    • Triplet: 3 bases per 1 amino acid
    • Universal: The same code is used in (almost) all organisms.
    • Non-ambiguous: A given codon always encodes the same amino acid.
    • Degenerate: More than one codon is possible for most amino acids (64 codons, 20 amino acids). This is accomplished primarily through "wobble" pairing at the third codon position. (Wobble pairing)(more)(more)
    • Non-overlapping Well??? What about overlapping genes.
    • Commaless (What about introns?)
*(From "Aminoacylation of tRNA 2′- or 3′-hydroxyl by phosphoseryl- and pyrrolysyl-tRNA synthetases," Published online 2013 Sep 8. doi:10.1016/j.febslet.2013.08.037 by Englert et al.):
   Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that catalyze the attachment of amino acids to corresponding tRNAs [1]. The resulting aminoacyl-tRNAs (AA-tRNAs) are transported by the elongation factor (EF-Tu in bacteria and EF1A in archaea and eukaryotes) to the ribosome as building blocks for protein synthesis [2,3]. AARSs catalyze formation of AA-tRNAs in two steps at the same active site: activation of the amino acid with ATP to form an aminoacyl-adenylate (AA-AMP), and transfer of the amino acid moiety to the 2′- or 3′-hydroxyl group (OH) of the tRNA terminal adenosine (A76) [1,4]. Based on the active site structure, AARSs are grouped into two independently evolved classes [5]. Class I AARSs all attach amino acids to the 2′-OH of A76, whereas most Class II enzymes aminoacylate the 3′-OH except asparaginyl- (AsnRS) and phenylalanyl-tRNA (PheRS) synthetases that prefer the 2′-OH [4,6,7].
   In solution, the 2′- or 3′-linked amino acid spontaneously transacylates to the neighboring OH at high rates [8], resulting in a mixture of 2′- and 3′-linked AA-tRNA isomers. EF-Tu stabilizes the 3′-isomer, which is preferred by the ribosome during peptide bond formation [9]. The vicinal hydroxyl group plays critical roles in catalyzing peptide bond formation on the ribosome, and hydrolyzing (editing) misacylated tRNAs by several AARSs and trans-editing factors [1013].

**(From Molecular Biology of the Cell, Alberts et al., 5th ed., Garland Science): "Most cells have a different synthetase enzyme for each amino acid (that is, 20 synthetases in all); one attaches glycine to all tRNAs that recognize codons for glycine, another attaches alanine to all tRNAs that recognize codons for alanine, and so on. Many bacteria, however, have fewer than 20 synthetases, and the same synthetase enzyme is responsible for coupling more than one amino acid to the appropriate tRNAs. In these cases, a single synthetase places the identical amino acid on two different types of tRNAs, only one of which has an anticodon that matches the amino acid. A second enzyme then chemically modifies each 'incorrectly' attached amino acid so that it now corresponds to the anticodon displayed by its covalently linked tRNA."
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