1. DNA, RNA, and Protein Chapter 2 Clark & Pazdernik

2. 2 The Central Dogma Cells store the genetic information to function and replicate in their DNA. When a protein is needed, DNA is transcribed into RNA, which in turn, is translated into a protein. FIGURE 2.1 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

3. 3 The Structure of a Typical Gene Genes are regions of DNA that are transcribed to give RNA. In most cases, the RNA is translated into protein, but some RNA is not. The gene has a promoter region plus transcriptional start and stop points that flank the actual message. After transcription, the RNA has a 5’ untranslated region (5’ UTR) and 3’ untranslated region (3’ UTR), which are not translated; only the ORF is translated into protein. FIGURE 2.2 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

4. 4 RNA Polymerase Synthesizes RNA at the Transcription Bubble RNA polymerase is a complex enzyme with two grooves. The first groove holds a single strand of DNA, and the second groove holds the growing RNA. RNA polymerase travels down the DNA, adding ribonucleotides that complement each of the bases on the DNA template strand. FIGURE 2.3 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

5. 5 Monocistronic versus Polycistronic Eukaryotes transcribe genes in single units, where each mRNA encodes for only one protein. Prokaryotes transcribe genes in operons as one single mRNA, and then translate the proteins as separate units. FIGURE 2.4 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

6. 6 Eukaryotic Transcription Many different general transcription factors help RNA polymerase II find the TATA and initiator box region of a eukaryotic promoter. FIGURE 2.5 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

7. 7 Components of the lac Operon The lac operon consists of three structural genes, lacZYA, which are all transcribed from a single promoter, designated lacP. The promoter is regulated by binding of the repressor at the operator, lacO, and of Crp protein at the Crp site. Note that in reality, the operator partly overlaps both the promoter and the lacZ structural gene. The single lac mRNA is translated to produce the LacZ, LacY, and LacA proteins. The lacI gene that encodes the LacI repressor has its own promoter and is transcribed in the opposite direction from the lacZYA operon. FIGURE 2.6 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

8. 8 Control of Lactose Operon The lactose operon is turned on only when glucose is absent but lactose is present. When glucose is available, the global activator protein, Crp, does not activate binding of RNA polymerase. When there is no glucose, Crp binds to the promoter and stimulates RNA polymerase to bind. The lack of lactose keeps LacI protein bound to the operator site and prevents RNA polymerase from transcribing the operon. Only when lactose is present is LacI released from the DNA. FIGURE 2.7 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

9. 9 Structures of Lactose, allo-Lactose, and IPTG IPTG is a nonmetabolizable analog of the lactose operon inducer, allo-lactose. β-galactosidase cannot break the sulfur linkage, and therefore, does not cleave IPTG in two. FIGURE 2.8 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

10. 10 Model of Two-Component Regulatory System The two-component regulatory system includes a membrane component (sensor kinase) and a cytoplasmic component (regulator). Outside the cell, the sensor domain of the kinase detects an environmental change, which leads to phosphorylation of the transmitter domain. The response regulator protein receives the phosphate group, and as a consequence, changes configuration so as to bind the DNA. FIGURE 2.9 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

11. 11 Transcription Factors Have Two Independent Domains (A) One domain of the GAL4 transcription factor normally binds to the GAL4 DNA recognition sequence and the other binds the transcription apparatus. (B) If the LexA sequence is substituted for the GAL4 site, the transcription factor does not recognize or bind the DNA. (C) An artificial protein made by combining a LexA binding domain with a GAL4 activator domain will not recognize the GAL4 site, but (D) will bind to the LexA recognition sequence and activate transcription. Thus, the GAL4 activator domain acts independently of any particular recognition sequence. It works as long as it is held in close contact with the DNA. FIGURE 2.10 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

12. 12 Somatic Mutations The early embryo has the same genetic information in every cell. During division of a somatic cell, a mutation may occur that affects the organ or tissue it gives rise to. Because the mutation was isolated in a single precursor cell, other parts of the body and the germline cells will not contain the mutation. Consequently, the mutation will not be passed on to any offspring. FIGURE 2.11 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

13. 13 Eukaryotic Regulation of Transcription (A) AP-1 is a eukaryotic transcription factor that consists of Fos and Jun. These two proteins interact through their leucine zippers. (B) To activate transcription, AP-1 must itself first be activated by phosphorylation by the kinase, JNK. Only then does Jun stimulate RNA polymerase II to transcribe the appropriate genes. FIGURE 2.12 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

14. 14 DNA Methylation Induces Gene Silencing Gene expression in eukaryotes can be turned off by chromatin condensation. First, the area to be silenced is methylated. The methyl groups attract methyl cytosine binding protein, which in turn attracts histone deacetylases. Once HDAC removes the acetyl groups from the histone tails, the histones aggregate tightly. The closeness of histones excludes any DNA binding proteins and hence turns off gene expression in the area. FIGURE 2.13 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

15. 15 Processing Eukaryotic mRNA Eukaryotic RNA is processed before exiting the nucleus for translation into protein. A guanine with a methyl group is added to the 5’ end of the message, a poly(A) tail is added to the 3’ end, and the introns are spliced out. These modifications stabilize the message and make it much shorter than the original RNA transcribed from the DNA. FIGURE 2.14 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

16. 16 The Genetic Code The 64 codons found in mRNA are shown with their corresponding amino acids. As usual, bases are read from 5’ to 3’ so that the first base is at the 5’ end of the codon. Three codons (UAA, UAG, UGA) have no cognate amino acid but signal stop. AUG (encoding methionine) and, much less often, GUG (encoding valine) act as start codons. To locate a codon, find the first base in the vertical column on the left, the second base in the horizontal row at the top, and the third base in the vertical column on the right. FIGURE 2.15 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

17. 17 Structure of tRNA Allows Wobble in the Third Position Transfer RNA recognizes the codons along mRNA and presents the correct amino acid for each codon. The first position of the anticodon on tRNA matches the third position of the codon. FIGURE 2.16 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

18. 18 Translation in Prokaryotes (A) Initiation of translation begins with the association of the small ribosome subunit with the Shine-Dalgarno sequence (S-D sequence) on the mRNA. Next, the initiator tRNA that reads AUG is charged with fMet. The charged initiator tRNA associates with the small ribosome subunit and finds the start codon. Assembly is helped by initiation factors (IF1, IF2, and IF3)—not shown. (B) During elongation peptide bonds are formed between the amino acids at the A-site and the P- site. The movement of the ribosome along the mRNA and addition of a new tRNA to the A-site are controlled by elongation factors (also not shown). (C) Termination requires release factors. The various components dissociate. The completed protein folds into its proper three-dimensional shape. FIGURE 2.17 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

19. 19 Translation in Eukaryotes (A) Assembly of the small subunit plus initiator Met-tRNA involves the binding of factors eIF3 and eIF2. (B) The cap binding protein of eIF4 attaches to the mRNA before it joins the small subunit. (C) The mRNA binds to the small subunit via cap binding protein and the 40S initiation complex is assembled. (D) Assembly of the large subunit requires factor eIF5. After assembly, eIF2 and eIF3 depart. FIGURE 2.18 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.

20. 20 Human Mitochondrial DNA The mitochondrial DNA of humans contains the genes for ribosomal RNA (16S and 12S), some transfer RNAs (single-letter amino acid codes mark these on the genome), and some proteins of the electron transport chain. FIGURE 2.19 Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.