This is old Lecture 23

Textbook Assignment: Chapter 12, Pages 347-363.

This is the last lecture in a series of four reviewing basic concepts of molecular biology and the central dogma that are covered in MCDB 1150.

Translation: major concepts: Translation is a polymerization process in which amino acids are joined together by means of peptide bonds to form proteins. The amino acid sequence of each protein is determined by the sequence of ribonucleotides in messenger RNA (mRNA), which in most cases has previously been transcribed from DNA. The amino acid sequence is specified by a nucleotide triplet code in the mRNA. That code is read by anticodons on transfer RNA (tRNA) molecules. Specific types of tRNA molecules are charged with specific amino acids by animoacyl tRNA synthetase molecules, which are the only "bilingual" component of the translation process. If a tRNA is charged with the wrong amino acid, that amino acid will be inserted into the protein in place of the amino acid that should have been on the tRNA. The joining of tRNAs to their respective amino acids is an energy-requiring process driven by hydrolysis of ATP, with an aminoacyl-AMP intermediate.

Translation occurs on ribosomes, which are assembled from subunits each time a new translation event is initiated. Hydrolysis of GTP is required for the binding of a charged aminoacyl-tRNA to the ribosome, and hydrolysis of a second GTP is required for a translocation process that prepares the ribosome to accept the next aminoacyl-tRNA. Termination of peptide chain growth occurs at specific codons in the message and also requires hydrolysis of a GTP. Details of translation and the major molecular species involved are summarized below in outline form, arranged in approximately the same sequence as in the textbook (figure 12.20). Except where specified otherwise, the descriptions are for E. coli, which is representative of typical prokaryotic cells. Eukaryotic translation is similar in principle, but differs in many of its details.

Linking amino acids to the appropriate tRNAs

• Transfer RNA (tRNA) molecules are about 75 nucleotides in length and fold by internal base pairing into a typical "cloverleaf" configuration, which in itself is a flattened distortion of the actual shape (see figures 12.17 and 12.18.
• There are about 50 different molecular species of tRNA in E. coli, able to pair selectively with 61 different codons.
• The wobble hypothesis explains how some tRNA anticodons can pair with more than one codon.
• tRNAs contain numerous modified ribonucleotide bases, which have been altered post-transcriptionally.
• Each aminoacyl-tRNA synthetase (tRNA charging enzyme) recognizes a specific amino acid and also specific properties of the tRNA molecules for that amino acid, which consist of more than just their anticodons.
• Hydrolysis of ATP results in formation of an intermediate aminoacyl-5'-AMP complex that is carried on the aminoacyl-tRNA synthetase enzyme molecule (fig. 12.19).
• All tRNAs have a ...CCA-OH sequence at their 3'-ends. An ester bond is formed between the carboxyl group of the amino acid and a free 2'- or 3'- hydroxyl group at the 3' end of the tRNA. Class I aminoacyl-tRNA synthetases attach the amino acid at the 2'-position and class II enzymes attach it at the 3'-position.

Formation of the initiation complex. (Figure 12.20)

• The small ribosomal subunit (30S) consists of a 16S rRNA plus 21 proteins (S1 ... S21).
• Initiation factor 3 (IF-3) binds to the 30S subunit.
• The mRNA initially attaches to the 30S ribosome plus IF-3. IF-1 is also involved, but its exact role is not as clear.
• N-formylmethionine (fMet) binds to a special iniator tRNA, whose recognition site is AUG (the normal methionine codon).
• Charged fMet-tRNA binds to initiation factor 2 (IF-2) and GTP
• The fMet-tRNA/IF-2/GTP complex and the 30S/IF-3/mRNA complexes join together to form the initiation complex. The fMet-tRNA recognizes an AUG initiation codon on the mRNA, located just downstream from a second recognition site known as the Shine-Delgarno sequence (consensus sequence AGGAGGU), which is believed to be complementary to the 3'-end of the 16S ribosomal RNA. IF-3 is released.

Formation of the 70S ribosome complex

• The large ribosomal subunit (50S) consists of 21S and 5S RNAs plus 34 proteins (L1 ... L34). The 50S subunit joins onto the initiation complex to form a 70S ribosome complex. This step is driven by hydrolysis of the GTP attached to the initiation complex. IF-1 and IF-2 also dissociate at this step. (Note that 50S + 30S combine to generate 70S. The designation "S" refers to a Svedberg unit, which is a measure of rate of sedimentation in a centrifugal field. Although S values are related to the overall weights of sedimenting particles, they are not linear measures of weight.)

A and P sites, recruitment of aminoacyl-tRNAs, elongation of the peptide chain.

• The 70S ribosome complex contains two sites for attachment of tRNAs and the amino acids (or peptide chains) that they carry. The P site is initially occupied by the fMet-tRNA, and becomes the attachment site for the growing peptide chain. The A site is the initial attachment site for tRNAs that bring new amino acids to the growing peptide chain.
• Elongation factor EF-Tu forms a complex with GTP.
• fMet-tRNA initially occupies the P site.
• A new charged tRNA attaches to the A site, guided by the codon-anticodon match between the mRNA and the tRNA, with the attachment driven by hydrolysis of the GTP attached to EF-Tu when the correct match is found.
• fMet (or the growing peptide chain in subsequent cycles) is transferred from the 3' end of the tRNA in the P site to the amino group of the amino acid in the A site, forming a new peptide bond.
• The empty tRNA is released from the P site.
• The tRNA with the growing peptide chain is then transferred from the A site to the P site. This is catalyzed by elongation factor G (EF-G) and driven by hydrolysis of another GTP. In the process, the position of the mRNA is shifted by one codon (3 bases), such that the codon for the next amino acid is now in the A site. GDP is displaced from EF-Tu by elongation factor Ts (EF-Ts). GTP then replaces EF-Ts, regenerating the EF-Tu/GTP complex.
• The charged tRNA bearing the next amino acid is recruited and the cycle is repeated.

Termination of translation

• New amino acids continue to be recruited and added to the growing peptide chain until a termination codon (UAG, UAA, UGA) enters the A position. Termination requires the participation of at least three release factors, RF-1, RF-2, and RF-3. RF-1 recognizes stop codons UAA and UAG, and RF-2 recognizes UAA and UGA. This leaves the finished peptide still attached at its carboxyl end to the tRNA in the P position. The completed protein is then released, catalyzed by RF-3 and driven by the hydrolysis of yet another GTP.
• IF-3 then binds the 30S subunit, causing dissociation of the 70S ribosome.

Energy consumed during protein synthesis.

• Formation of aminoacyl-tRNA complex requires hydrolysis of ATP to AMP plus pyrophosphate. Subsequent hydrolysis of pyrophosphate helps "pull" the reaction forward by mass action.
• Two GTPs are hydrolyzed to GDP, one during attachement of aminoacyl-tRNA to the A site, and one during the translocation step.
• The "cost" to the cell of adding one amino acid to a growing polypeptide chain is four phosphate bonds, which directly describes the amount of work that the cell must do to regenerate one ATP from AMP and two GTPs from GDPs. In terms of the actual amount of energy put into the synthetic process, three bonds are directly hydrolyzed (ATP --> AMP and 2 GTP --> 2 GMP). However, because the ATP-driven reaction is also pulled forward by hydrolysis of pyrophosphate that is derived from the ATP, it is probably more correct to say that four high energy bonds are hydrolyzed in order to add one amino acid to a growing peptide chain. The previous text used in this course estimated that 90% of the total energy production of an E. coli cell goes into protein synthesis.

Post-translational modification of proteins.

• Protein folding and post-translational modification, which are described in the last part of chapter 13 (pages 378-383), were discussed briefly in lecture 20.
• We do not have time to explore these phenomena in detail in this class, but it is important to recognize that many additional modifications are generally needed after translation to generate the final functional protein product. These include processes such as glycosylation, phosphorylation, formation of disulfide bonds, and modification of amino acids, as well as complex folding and often associations between two or more separate polypeptide chains.

New material: This lecture marks the end of the brief review of material from MCDB 1150. Beginning with the next lecture we will return to our previous pattern of examining new material in greater detail than has been possible in these "review" lectures.

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