MCB 201 Gene Expression - Spring Semester 2004
Lecture 4 (Synthesis of Biopolymers in General and Nucleic Acids in Particular)
1. Figure 4-1, Lodish5e: The basic genetic processes. This is a good summary for the basic processes of information transfer in cells. This diagram is based on the original Central Dogma of information transfer proposed by Francis Crick in the 1950's. It doesn't show an arrow back from RNA to DNA to symbolize reverse transcription, which was discovered in the late 1960's, since it just emphasizes the basic pathways common to all cells.
2. Synthesis of Biopolymers: rules of macromolecular carpentry, four rules that apply to the synthesis of both proteins and nucleic acids:
- a. Proteins and nucleic acids are built from a relatively small number of different monomeric building blocks, the 20 naturally occurring amino acids for proteins, and 5 nitrogen bases attached to either a common deoxyribose phosphate structure (DNA) or a ribose phosphate structure (RNA) for nucleic acids.
- b. The monomers are added one at a time.
- c. Each polypeptide and polynucleotide chain has a specific starting point, and growth proceeds in one direction to a fixed terminus, for proteins the direction is N-terminus to C-terminus, and for nucleic acids the direction is 5' to 3'.
- d. The primary synthetic product is often modified.
3. Figure 4-13, Lodish4e: Chain elongation in the in vivo synthesis of both proteins and nucleic acids proceeds by sequential addition of monomeric units - amino acids and nucleotides, respectively. Note the 'shorthand' structures used in this figure to represent nucleic acid chains. Go back to figure 4-2 Lodish5e to see the relationship of this notation to the chemical structures. Then, return to figure 4-13 Lodish4e. This figure emphasizes the directionality of chain elongation for both polypeptides and nucleic acids. Peptide bond formation during protein synthesis is a dehydration reaction, which requires input of energy; therefore it is not thermodynamically favorable (spontaneous). You may recall from biochemistry that hydrolysis reactions, which release energy, are favored thermodynamically. Likewise, phosphodiester bond formation during nucleic acid synthesis is energy requiring. Note that one the products pyrophosphate goes on to be hydrolyzed, releasing energy and making the overall reaction more favorable thermodynamically.
4. Figure 4-14, Lodish4e: Proteins and nucleic acids typically undergo one or more modifications after the basic polymeric chains are formed. As we will learn in more detail later, DNA undergoes cleavage of phosphodiester bonds by Dnase enzymes, e.g. restriction endonucleases, and these bonds can be reformed by DNA ligase during DNA replication, repair and recombination. RNA produced during transcription may undergo splicing reactions which again involves breaking and reforming phosphodiester bonds. Recently, self-splicing has been discovered in proteins whereby pieces of polypeptide chain called inteins are removed by proteases and the peptide bond resealed. Chemical crosslinking rarely occurs between nucleic acid chains. If this happens in DNA it may cause a mutation event. Crosslinking occurs more commonly in proteins. Both nucleic acids and proteins can undergo chemical modifications to functional groups. For example serine and threonine residues in proteins may be phosphorylated. Methylation of certain nitrogen bases in DNA is common.
Section 4.2 (Transcription of protein-coding genes and formation of functional mRNA).
5. We start with a general overview of the concepts involved in nucleic acid synthesis in cells.
6. The syntheses of DNA and RNA have certain features in common and other aspects are unique to each process.
The common features include:
a. New chains are produced using an existing or parental strand as a template. The concept of templates in information transfer is extremely important. This is how the hard-won biological wisdom of billions of years of evolution is preserved and transmitted to progeny. The new strand that is produced is not chemically identical to the parental template. It is related to the template strand by Chargaff's rules of complementary base-pairing, so it is said to be complementary to the template strand.
b. Synthesis proceeds in the 5' to 3' direction. Synthesis requires a supply of 5' triphosphates of ribonucleosides for RNA synthesis and 5' triphosphates of deoxyribonucleosides for DNA synthesis. Like the intermediate energy carrier ATP with which you are already familiar, these compounds are relatively energy-rich and provide the energy to produce the phosphodiester bonds during new chain synthesis.
7. Figure 4-9, Lodish5e. A simplified diagram of the process of transcription or RNA synthesis is shown here. Notice how the parental strand shown in blue provides the template and that the base-pairing rules are followed (GC, AU). RNA polymerase is the enzyme that forms the phosphodiester bonds which are formed by a reaction between the 3'-OH of the growing RNA strand and the alpha phosphate group of the incoming ribonucleotide (rNTP), as called a ribonucleoside triphosphate. RNA strands are synthesized in the 5' to 3' direction and are antiparallel to their template DNA strand.
8. Figure 4-10, Lodish5e: Three stages in transcription:
A. Initiation. RNA polymerase binds to its promoter sequence, usually the TATA box in double helical, as called duplex, DNA (called the "closed complex" at this stage) and as RNA synthesis begins, a transcription bubble forms. This bubble is called the "open complex". The RNA polymerase catalyzes the linkage by phosphodiester bonding of the two initial ribonuleotides.
B. Elongation. The newly started RNA strand, complementary in base sequence to the DNA template strand, is called a nascent RNA strand. The polymerase moves down the template DNA strand, moving in the 3' to 5' direction on the DNA template, and adds on ribonucleotide at a time to the nascent RNA chain. The Chargaff rules of base pairing are followed, with U being used in the RNA chain to pair with A. In DNA, this pair would be A-T.
C. Termination. RNA synthesis ends when the RNA polymerases moves onto a specific termination or transcriptional stop sequence on the DNA template. At this point, the polymerase releases the completed RNA strand and dissociates from the DNA template.
9. Figure 4-11, Lodish5e: Current model of bacterial RNA polymerase bound to a promoter. The five protein subunits of the core polymerase are shown in colors. The DNA template strand is shown in gray and the nontemplate DNA strand is colored pink. The catalytic center is marked by a gray sphere representing a Mg++ ion. Note that the DNA is bend sharply in this complex, which corresponds to step 2, i.e. the open complex, in Figure 4-10, Lodish5e.
Media Connections: Focus Animation: Basic Transcriptional Mechanism
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