Revised September 3, 1999. This lecture is based on 1998 Lecture 24. Additional material from the draft of lecture 9 was added at the end of these notes on Septemer 11, 1999. A copy was retained also in lecture 9 to make sure that everyone sees it.

Lecture 6, MCDB 2150 Fall 1999

Review of Ribosomes and Translation

Textbook Assignment: Chapter 4, Pages 88-123.

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

Overview: Preparation for translation: 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.

Overview: Assembly of peptide chains: 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 each 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 stop 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 (see figures 4.8, 4.14, 4.24, 4.25 and 4.26 for a visual summary of the main steps). 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.

Ribosomes: Ribosomes, which provide the sites at which protein synthesis are complex molecular aggregates, composed of large and small subunits, which assemble together only for the purpose of protein synthesis and separate again when the process is completed. The ribosomes of E. coli consist of a small subunit containing one 16S RNA and 21 different proteins plus a large subunit containing two RNAs (23S and 5S) plus 31 different proteins (figure 4.8). (Note that most textbooks claim there are 34 proteins in the large subunit -- I have not yet determined whether this in an error in our book or new information, but for our purposes, the difference is trivial). Mammalian ribosomes consist of a small subunit containing an 18S RNA and 30-35 proteins, plus a large subunit with 28S, 5.8S, and 5S RNAs and 45-50 different proteins (figure 4.9). The abbreviation "S" refers to Svedberg Units, a measurement of the rate of sedimentation during high speed centrifugation, which reflects the relative sizes on the RNA molecules, but not in a strictly linear relationship. As shown in figure 4.10, ribosomes attach to messenger RNA and synthesize proteins as they progress from one end of the coding sequence to the other. Many ribosomes can be seen attached to a single actively translated mRNA, forming a complex known as a polysome.

Linking amino acids to the appropriate tRNAs

Formation of the prokaryotic initiation complex. (Figure 4.22)

Formation of the 70S ribosome complex (Figure 4.25d)

A and P sites, recruitment of aminoacyl-tRNAs, elongation (Figure 4.25)

Termination of translation

Eukaryotic translation

Energy consumed during protein synthesis.

Post-translational modification of proteins.

The material that follows was moved to this location from the draft of lecture 9 on September 11, 1999. It covers material from the end of Chapter 4 that is briefly summarized in the previous paragraph. It also introduces the concept of prions as self-duplicating abnormally folded proteins, a topic that is not covered in our textbook. A copy has also been retained in lecture 9 for this year.

Protein structure: Section 4.9 on protein structure and function should be read as additional bakcground information that is essentially a review of material from MCDB 1150. Four levels of structural information are commonly recognized.

Post-translational modification: Proteins are subject to a variety of post-translational modifications, including frequent removal of N-terminal methionine, removal of other N- or C- terminal sequences, removal of internal sequences, removal of signal or targeting sequences, modification of specific amino acids (such as conversion of proline to hydroxyproline), phosphorylation of hydroxyl groups, addition of carbohydrate side chains (glycosylation), complexing with metals or other prosthetic groups, and a long list of other possibilities that are not discussed particularly well in the textbook. Some of these modifications are illustrated in a section on enzymes and enzyme activity at the end of the chapter.

Prions: A boxed section at the end of chapter 13 of Klug and Cummings, Concepts of Genetics, 5th edition (the previous text for this course, available at the Norlin reserve desk) discusses an unusual pathogenic unit called a prion (proteinaceous infective agent). Although the prion theory remains controversial, a very large amount of evidence has accumulated showing that prion proteins are coded by the host, and subsequently modified to function as pathogens. The modified proteins accumulate in aggregates that cause degenerative diseases of the brain. The best available evidence seems to indicate that a conformational modification of the normal host protein gives it pathogenic properties plus the ability to catalyze similar modification of additional normal proteins, such that the pathology is infectious. The most current focus on prions is the mad cow disease , which apparently got its start when proteins derived from sheep infected with a similar disease, scrapie, were used in cattle feed. Ordinary sterilization techniques do not inactivate the prion infectivity. Similar diseases are known in humans, including Kuru and Creutzfeld-Jacob disease. There may also have been some cases of animal to human transmission, although these have not been positively verified. Stanley Prusiner was awarded the Nobel Prize in 1998 for his work on prions.

New material ahead! This lecture marks the end of the brief "review" of material from MCDB 1150. Starting with the next lecture we will begin to examine "new" material in greater detail than has been possible in these "review" lectures.