© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION Gene expression and regulation 4 David G. Bear Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, NM TRANSLATION OF A MESSENGER RNA (mRNA) isolated from a cell in the salivary gland of an insect Chironomous tentans in this digitally colored trans- mission electron micrograph. Translation is the pro- cess by which ribosomes (blue particles) translocate along the mRNA (pink strand) and read its sequence in three nucleotide units called codons. As the ribo- some moves to each codon on the mRNA, it inserts the appropriate amino acid into the growing protein chain (green filaments). (© Dr. Elena Kiseleva/SPL/ Photo Researchers, Inc.) CHAPTER OUTLINE 4.1 Introduction 4.12 Translation is a three-stage process that decodes an 4.2 Genes are transcription units mRNA to synthesize a protein 4.3 Transcription is a multistep process directed by 4.13 Translation is catalyzed by the ribosome DNA-dependent RNA polymerase 4.14 Translation is guided by a large number of protein 4.4 RNA polymerases are large multisubunit protein factors that regulate the interaction of amino- complexes acylated tRNAs with the ribosome 4.5 Promoters direct the initiation of transcription 4.15 Translation is controlled by the interaction of the 4.6 Activators and repressors regulate transcription 5′ and 3′ ends of the mRNA and by translational initiation repressor proteins 4.7 Transcriptional regulatory circuits control eukaryotic 4.16 Some mRNAs are translated at specific locations cell growth, proliferation, and differentiation within the cytoplasm 4.17 Sequence elements in the 5 and 3 untranslated 4.8 The 5′ and 3′ ends of mature mRNAs are generated ′ ′ by RNA processing regions determine the stability of an mRNA 4.9 Terminators direct the end of transcription elongation 4.18 Noncoding RNAs are important regulators of gene expression 4.10 Introns in eukaryotic pre-mRNAs are removed by the spliceosome 4.19 What’s next? 4.11 Alternative splicing generates protein diversity 4.20 Summary References 2ND PAGES 9781284023558_CH04_0103.indd 103 6/11/13 4:20 PM © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION 4.1 Introduction DNA Key concepts Replication • DNA functions as the template for the synthesis of Reverse Transcription RNA by RNA polymerase (transcription). Messenger transcription RNA (mRNA) directs the synthesis of proteins by the ribosome (translation). • The genetic code refers to the set of 64 base RNA triplets (codons) that are read by the ribosome Replication during translation. Translation • Each of the 61 codons codes for 1 of the 20 common amino acids, whereas 3 codons cause the ribosome to stop translation. Protein • In prokaryotes, transcription and translation of mRNA occur concomitantly in the same region of the cell. • In eukaryotes, transcription and mRNA processing occur concomitantly inside the nucleus, whereas FIGURE 4.1 The Central Dogma of Molecular Biology. The translation occurs subsequent to the export of the Central Dogma states that DNA serves as the template mRNA to the cytoplasm. for the synthesis of RNA by the enzyme RNA polymerase (transcription) and the RNA transcript is decoded by the Modern biology began in the early part of the ribosome to synthesize a polypeptide (translation). The 20th century with the direct examination of the enzyme reverse transcriptase can synthesize DNA from structure and function of cells, first by improved RNA. Reverse transcription is a rare event in normal cells. microscopy techniques that permitted the de- Many RNA viruses contain a reverse-transcriptase gene tailed study of cell structure and then by high- in their genome. speed centrifugation methods that facilitated biochemical isolation and characterization of mechanisms of transcription and translation, cytoplasmic and nuclear subcellular fractions. with details of the structural and functional Geneticists focused on the relationship between properties of RNA polymerases that tran- the dynamic structure of chromosomes within scribe the genome and ribosomes that trans- the cell nucleus and the mechanisms of genetic late mRNA to make protein. This work also inheritance, which culminated with the eluci- revealed that transcription and translation are dation of the structures of DNA and RNA and an coupled in bacteria—the mRNA is translated understanding of the genetic code. The scientific concomitantly as the newly synthesized RNA revolution of molecular biology that took place emerges from the active site of RNA poly- in the mid-20th century provided a framework merase (FIGURE 4.2). known as the Central Dogma of Molecular Biology: DNA functions as the template for the synthesis of RNA (transcription) and RNA directs the synthesis of protein by ribosomes Transcription (translation), as depicted in FIGURE 4.1. Initially, molecular biology focused on reconciling the classical concept of the gene— the unit of information controlling inherited DNA traits—with the more modern tenets of the RNA Central Dogma. These studies led to the One Gene-One Protein Hypothesis that posited Translation a gene to be the unit of genetic information that directs the synthesis of a specific mRNA, which in turn encodes an individual protein. Much of the early work on the Central Dogma Ribosome translates mRNA was carried out using a nonpathogenic strain of the bacterium Escherichia coli because of the ease of genetic manipulation and the ability to FIGURE 4.2 Gene expression in prokaryotes. Prokaryotes easily fractionate the bacterial cellular compo- do not have a cell nucleus. Thus, transcription and trans- nents for biochemical analysis. Studies with lation take place in the same cellular compartment and E. coli provided our first understanding of the occur concomitantly. 104 CHAPTER 4 Gene expression and regulation 2ND PAGES 2ND PAGES 9781284023558_CH04_0103.indd 104 6/11/13 4:21 PM © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION In the latter half of the 20th century, in- In addition to splicing, eukaryotic mRNAs un- vestigations of gene expression in several eu- dergo other posttranscriptional modifications, karyotic model systems, including the yeast including the addition of a 7-methylguanosine Saccharomyces cerevisiae, the insect Drosophila (7-MeG) cap to the 5′ end and a poly(A) tail melanogaster, and the human HeLa cultured cell comprising 100 to 300 adenosine ribonucleo- line, showed that many fundamental mecha- tides on the 3′ end. Selected mRNAs can also nisms of gene expression are conserved among have additional bases added posttranscription- all organisms; however, there are some basic ally in a process referred to as mRNA editing. differences between prokaryotes and eukary- Analogous to transcription–translation coupling otes, particularly with regard to the posttran- in prokaryotes, most eukaryotic mRNA process- scriptional pathways of mRNA biosynthesis ing reactions are coupled to transcription. (FIGURE 4.3). Some of the greatest similarities, as well as One of the most surprising discoveries was some of the biggest differences, between pro- that most protein-coding genes in higher eukary- karyotic and eukaryotic cells are at the final step otes are interrupted with DNA segments, termed of gene expression—translation. Prokaryotes introns, that are transcribed into RNA but are re- lack a cell nucleus, and the translation complex moved before translation. Ribonucleoprotein (ribosomes and associated factors) is in direct (RNP) complexes, referred to as spliceosomes, contact with the transcription apparatus on the were found to excise the introns from the orig- chromosomal DNA, whereas in eukaryotes the inal mRNA, or pre- mRNA, and to ligate the transcription and RNA processing reactions oc- remaining expressed mRNA sequences, termed cur in the nucleus and translation occurs in exons, together to form the mature mRNA. the cytoplasm. This separation of RNA biosyn- thesis from translation necessitates the pack- aging of mRNA into an mRNA RNP (mRNP) complex that then must be exported through Exon 1 Exon 2 the pores of the nuclear envelope into the cy- DNA Intron toplasm before translation. In spite of the extra steps between transcription and translation in eukaryotes, the prokaryotic and eukaryotic translational machineries are very similar. In fact, some of the highest evolutionary conser- Transcription vation of macromolecular structure and func- Nucleus tion among species is found in ribosomes and associated translational factors. 5 Intron The information contained in a mature 3 Pre-mRNA mRNA for making a protein is decoded by the ribosome. The ribosome reads the mRNA se- Intron Splicing quence in three-nucleotide units referred to 5 as codons. Because there are four different 3 nucleotide bases in DNA and RNA—A, G, C, mRNA and T (U in RNA)—there are 64 possible codons (FIGURE 4.4). Each codon specifies the insertion Transport by the ribosome of a specific amino acid into tRNA the protein. Twenty amino acids are commonly 5 3 found in proteins; thus, some amino acids are specified by more than one codon. In general, Ribosome codons that encode the same amino acid differ Translation Cytoplasm by only one base. The tendency for amino acids with chemically similar functional groups to be represented by related codons minimizes the effects of mutations by increasing the like- Protein lihood that a single random base change will FIGURE 4.3 Gene expression in eukaryotes. Eukaryotes result in an alternative amino acid substitu- contain a cell nucleus where transcription and RNA pro- tion with similar molecular characteristics. For cessing occur. The mRNA transcript is exported from the example, a mutation of CUC to CUG has no nucleus to the cytoplasm where translation takes place. effect, because both codons represent leucine. 4.1 Introduction 105 2ND PAGES 2ND PAGES 9781284023558_CH04_0103.indd 105 6/11/13 4:21 PM © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION cases, the first codon that specifies a protein First Position Second Position Third Position (5’-end) (3’-end) sequence is AUG, which specifies the amino acid methionine and is referred to as the start U C A G codon.