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Transcription and Translation
 

Transcription and Translation

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  • Archibald Garrod was one of the first scientists to propose that inherited factors (genes) controlled the function of proteins. Defects (diseases) in metabolism could be linked to the failure of specific enzymes to catalyze essential biochemical reactions. Since these disease phenotypes were passed from generation to generation in a regular pattern, the genes responsible for the appearance of the disease phenotype must somehow be controlling the function of the enzymes. So, the question that biologists had to tackle was how these inherited factors, genes, could control the structure and activity of enzymes (proteins). Garrod’s insight in 1908 that hereditary metabolic diseases (inborn errors of metabolism) resulted from inherited defects in an enzyme framed the question that biologists, chemists and physicists would spend the latter half of the 20th century answering.
  • One of the seminal papers in biology that addressed the relationship between genes and proteins was Beadle and Tatum’s 1941 paper in the Proceedings of the National Academy of Sciences “Genetic control of biochemical reactions in Neurospora ”. Many other studies had been conducted to determine the biochemical and genetic basis of known phenotypes, and those studies had established that many biochemical reactions were controlled by genes. Instead of investigating already observed phenotypes, Beadle and Tatum set out to induce mutations in genes and see what biochemical pathways were affected. They used the Neurospora model genetic system. Their study had several assumptions: 1) x-ray treatment of spores will induce heritable mutations in the genes, 2) lethal mutations in biochemical pathways will be detected by the inability of the organism to survive on minimal medium, 3) mutations that are lethal for an organism on minimal medium can be overcome by supplementing the medium (“complete medium”) with the required nutrient and the required nutrient can pass through cell walls and membranes to be used by the organism. They treated single-spore cultures growing on complete medium with x-rays to induce mutations. These cultures were then transferred to a “minimal” medium that requires the organism to perform all of the necessary biochemical reactions for survival. The minimal medium used only contained agar, salts, biotin, and a carbon source (disacharide, fat or other complex molecule). Organisms that could grow on complete medium but not minimal medium were “tested” on minimal medium supplemented with a specific nutrient (e.g., a known vitamin, amino acid, or glucose). Beadle and Tatum’s team x-rayed single-ascospore strains of Neurospora crassa or Neurospora sitophila . In this paper they report isolating three mutants that grow normally on complete medium but do not grow well at all on minimal medium with sucrose as the carbon source. One of the strains was unable to synthesize vitamin B6; the second strain was unable to synthesize the thiazole portion of vitamin B1, and the third strain was unable to synthesize p -aminobenzoic acid. They present details of the vitamin B6-deficient mutant. In their discussion of their results, Beadle and Tatum point out that this technique of inducing mutations that affect a known pathway will allow geneticists to determine if more than one gene is required for carrying out a given step in a biochemical pathways. They also suggest that this method may also allow the discover of additional required nutrients and compounds. Link to the Beadle and Tatum paper: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16588492
  • Although Beadle and Tatum proposed a one gene-one enzyme hypothesis in 1941, the evidence to support the hypothesis that genes controlled the structure and function of proteins could not be proven until much later when scientists understood more about the nature of DNA and its role in the cell as the genetic material. As early as 1957, Ingram showed that the two beta-chain subunits of hemoglobin from sickle-cell anemia patients (Hb S) contained a single-amino acid substitution of a valine for a glutamate at position six in the polypeptide chain (Ingram, V. M. 1957. Gene mutations in human haemoglobin: The chemical difference between normal and sickle cell haemoglobin. Nature . 180 , 326). Later this inherited single-amino acid change was shown to be responsible for the pleiotrophic phenotypes associated with sickle-cell anemia. In 1967 Murayama suggested that the replacement of the glutamate with the valine at the position six in the chain allowed the formation of hydrophobic bonding between the valine at position one and the substituted valine at position six. This hydrophobic bonding would produce a sticky key-like projection that could interact with other hemoglobin molecules allowing them to stick together or stack (Murayama, M. 1967. Structure of the sickle cell hemoglobin and molecular mechanism of the sickling phenomenon. Clinical Chemistry 14 , 578–588). Studies of the trypthophan synthase gene ( trpA ) in E. coli first indicated that the sequence of nucleotides in a gene determines the sequence of amino acids in a polypeptide.
  • So the evidence existed that there is colinearity between genes and proteins in bacteria—the sequence of bases in the DNA molecule determines the sequence of amino acids in the encoded protein. Now the question became, how does the information in the DNA molecule direct the synthesis of the protein? The answer to this question is the process of transcription: the sequence of nucleotides in the DNA molecule is recopied or transcribed into a corresponding sequence of bases in an RNA molecule. This RNA molecule (messenger RNA, mRNA) transports the genetic information from the chromosome to the cellular machinery that synthesizes protein.
  • How was the process of transcription elucidated? Evidence existed that indicated that proteins were assembled away from DNA on ribonucleic acid complexes called ribosomes. There were two theories about how genes worked. One theory suggested that each gene encoded a unique ribosome which would then synthesize a specific protein. The competing theory suggested that all ribosomes were essentially similarly constructed “protein factories” and that some sort of messenger molecule carried the genetic instructions for the synthesis of a particular protein from the DNA chromosome to the ribosome.
  • In an attempt to answer this question Brenner, Jacob and Messelson looked at the way that bacteriophage (bacterial viruses) infected E. coli and directed the production of phage proteins (Brenner, S., Jacob, F., Meselson, M. 1961. An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature . 190, 576-581). When E. coli cells are infected with phage T4, they stop making bacterial proteins and begin making phage proteins. Brenner et al. wanted to see what kinds of molecules had to be synthesized in order for this “switch” to occur. Did the bacterial cells have to make new nucleic acids in order to produce the phage proteins? Or, did they have to make new ribosomes that could direct the production of the phage proteins? To ask this question, they grew E. coli cultures in media that contained “heavy” nitrogen and carbon. When these “heavy” atoms are incorporated into macromolecules such as proteins or nucleic acids, the macromolecules will be more dense and therefore migrate differently through a density gradient than macromolecules that only contained “normal” or “light” carbon and nitrogen atoms. They infected the cells with the heavy molecules with phage T4 and immediately transferred the cells to media containing “light” carbon and nitrogen atoms and the radioactive base uracil, which is a component found in RNA but not DNA.
  • If the hypothesis of a messenger molecule is correct, then the prediction would be that no new ribosomes would be synthesized (they would remain “heavy”). Furthermore if the messenger molecule was some type of RNA, it would contain the radioactively labeled uracil. The phage-infected cells did not make new “light” ribosomes, indicating that the same ribosomes were used to synthesize both phage and bacterial proteins. The specificity for protein synthesis had to be conferred by some type of messenger molecule. In this system the researchers were able to detect newly synthesized RNA molecules that contained the labeled uracil, and that RNA was associated with the ribosomes. Additionally, when the RNA was incubated with bacterial and phage DNA, it formed complexes (hybridized) with the phage DNA.
  • The process of gene transcription involves several steps and requires many proteins and other molecules. Here we will discuss the basics of transcription. RNA is composed of the nitrogenous bases adenine, guanine, cytosine and uracil, a ribose sugar, and phosphates. The sugar and the phosphates form the backbone of the RNA molecule, as they do in DNA. The ribonucleoside 5´ triphosphates required for RNA synthesis are adenine triphosphate, guanine triphosphate, cytosine triphosphate and uracil triphosphate. Synthesis of an RNA strand requires a DNA-dependent RNA polymerase. The DNA-dependent RNA polymerase does not require a primer to begin synthesis, but it must have a DNA template. When referring to the synthesis of a primary RNA transcript to direct the synthesis of proteins, the DNA strand of the double helix that serves as the template for the RNA molecule is called the “sense” or “coding” strand. The opposite strand is the “noncoding” strand. The RNA molecule is synthesized as one long molecule in the 5´ to 3´ direction.
  • RNA polymerase II in eukaryotes is composed of several subunits and requires several accessory proteins in order to initiate and carryout transcription. RNA pol II is has twelve subunits, by contrast the bacterial RNA polymerase isolated from E. coli has five. The accessory proteins that RNA pol II requires are known as transcription factors. Transcription factors that are required for transcription of any gene are basal transcription factors and are usually named “TFII” followed by a letter. Basal transcription factors are conserved across eukaryotic species. RNA polymerase II activity is DNA-dependent, meaning that it must have a DNA template molecule before it can synthesize the RNA transcript. (There are template-indpendent polymerases, which have been particularly useful in the laboratory for determining the genetic code). The DNA-dependent polymerase must also have Mg 2+ and ribonucleoside 5´ triphosphates in order to carry out RNA synthesis. The RNA polymerase creates the new RNA strand from 5´ to 3’. RNA polymerase I in eukaryotes synthesizes “preribosomal” RNA. This RNA molecule contains the information for the 18S, 5.8S and 28S RNA molecules that are associated with the ribosome (rRNAs). RNA polymerase III in eukaryotes synthesizes the tRNAs, 5S RNA and other small nuclear RNAs. Eukaryotic RNA pol II can be inhibited by certain compounds including the antibiotic actinomycin D. Actinomycin D inhibits transcription by inserting into G-C rich regions of DNA molecules, causing the polymerase to stall because it cannot move along the DNA template.
  • Transcription can be arbitrarily divided into several stages: promoter recognition (formation of closed, then open complex), chain initiation (incorporation of the first few bases), chain elongation and chain termination.
  • The closed complex of transcription (eukaryotic) is formed in the following steps: The TATA-binding protein (TBP) binds to the TATA box. TPB is bound by TFIIB, which also binds to the DNA on either side of the TBP. The TFIIB-TBP complex is bound by another complex consisting of TFIIF and RNA pol II. TFIIE and H bind to complete the closed complex. TFIIH has a helicase activity that can unwind the DNA around the transcription start site (+1).
  • Enhancers are DNA sequences that can control efficiency and rate of transcription. They do not have to be close to the gene promoter that they regulate, but they must be located on the same chromosome. Enhancers regulate the expression of a gene in specific cell types, and they control the timing of gene expression. Enhancers are critical for normal development because they regulate the timing and tissue-specificity of gene expression. Reporter genes are artificial gene expression systems that can be used to identify regions in DNA that are important for regulating gene expression. Basically the DNA promoter or enhancer region of interest is placed upstream of the reporter gene in a plasmid. This plasmid is then transformed into bacterial cells or transfected into eukaryotic cells. Activation of reporter gene expression results in some kind of detectable signal. Common reporter genes include beta-galactosidase, green fluorescent protein and luciferase. For instance, when a luciferase reporter gene is being actively transcribed, light will be produced when the substrate for luciferase is added to your cells or extract. To identify areas of DNA that are important for gene expression, deletions of increasing length are made in the attached promoter or enhancer region, and changes in signal as a result of gene expression or silencing are measured.
  • Transcription and translation in bacterial cells are coupled. As soon as enough of the primary transcript is synthesized to allow ribosome binding, translation begins, even if the polymerase is not finished synthesizing the transcript. In eukaryotic cells, however, the primary RNA transcript must be processed and transported to the cytoplasm before translation (protein synthesis) can begin. The DNA of eukaroytic genes contains noncoding sequences (introns) and coding sequences (exons). Introns must be removed from the RNA transcript by a process called splicing. Additionally a methylguanoisine cap is added to the 5´ end of the transcript, and a polyadenosine tail (polyA) is added to the 3´ end. The 5´ cap aids in transport of the completed mRNA from the nucleus, and the polyA tail helps to determine the stability of the mRNA molecule.
  • Some genes can produce several variants of the same protein by alternative splicing. For instance, the huge diversity of antibodies produced by the cells of the immune system results from the varied arrangements of “variable” (V), “diversity” (D) and “joining” (J) regions that are combined to make the final light and heavy chains within an antibody. Some of the VJ and VD rearrangements occur as a result of DNA splicing events that occur during B-cell differentiation as the germ-line DNA is rearranged to become the specialized DNA of the B-cell. Other RNA splicing events occur after the B-cell has differentiated after the RNA transcript is produced and is processed. Alternative splicing is an important biological phenomenon during development. Many RNAs are transcribed from either an adult or larval (juvenile or fetal) promoter. Alternative splicing can also produce tissue-specific proteins (for instance mouse liver and salivary amylase mRNAs are produced by alternative splicing events). Drosophila myosin RNA can occur in four forms, depending on the stage of development of the fly. This web site illustrates the alternative splicing of Drosophila DSCAM, axon guidance receptors. The alternative forms of these receptors that can be produced by alternative splicing contribute to a great diversity of receptor proteins and may play a role in the formation of complex neural circuits. Mutations at splice sites can also result in aberrant or truncated proteins that do not function properly. This figure illustrates the consequences of splice-site mutation: the failure to remove an intron, failure to include an exon, or activation of a cryptic splice site within the RNA molecule. Splice site mutations are responsible for a variety of human genetic disease. For instance, a common mutation observed in individuals suffering from thalassemia, is a splice-mutation in the beta-globin gene. Several pathogenic alleles of the breast cancer gene (BRCA 1) have been shown to be splice-site mutations.
  • The primary RNA transcript contains introns with donor splice sites at their 5´ ends and acceptor splice sites at their 3´ ends. In the middle of the intron is a branch site, usually an adenosine residue. The first step of the splicing process involves the 2´-OH of the adenosine at the branch site on the donor splice site, forming a 2´-5´ linkage and a loop or lariat structure, cleaving the 5´ intron/exon junction. The 5´ exon is placed close to the site of the second cleavage at the 3´ exon, and second cut is made. The 5´ and 3´ exons are joined. The region containing the lariat (the intron) is degraded. The first example of an RNA molecule that can catalyze a chemical reaction came from studies of splicing in Tetrahymena . Ribosomal RNA precursor self-splices. This is an example of something other than protein (RNA) catalyzing a chemical reaction.
  • Transcription gets the genetic information from the DNA out into the cytoplasm where new proteins are synthesized, but how is the information in the mRNA molecule used to direct the assembly of amino acids into a protein? The primary structure of a protein is the number and order of amino acids; there are 20 amino acids that can be found in proteins, but there are only four nitrogenous bases used in RNA. How do the four bases of RNA specify twenty amino acids? And, how are proteins started and stopped?
  • A simple math test can provide a clue about the nature of the genetic code. If you have 4 bases and combinations of 2 of them are required to specify an amino acid, then 4 squared is 16, but there are 20 amino acids. So, could they each be specified by a combination of three bases (4 cubed = 64). The sixty four possibilities created by 3-base “words” would clearly allow the code to specify all of the required amino acids? But what about all of the “extra” combinations? Are they just not used, or is the code redundant, with more than one 3-base word specifying the same amino acid? How does the code work? What are the start and stop signals that mark the beginning and end of a polypeptide chain? Is the code overlapping, with the 3-base words running on top of each other, or is the RNA message read 3 bases at a time? Francis Crick and colleagues published a paper in Nature in 1961 that set out arguments for a nonoverlapping, 3-base code. Their argument that the code is nonoverlapping is based on work by other groups (Wittmann; Tsugita and Fraenkel-Conrat) on tobacco mosaic virus. These groups showed that in TMV when the RNA is treated with nitrous acid, usually only one amino acid at a time is altered. If the code was overlapping, they would have predicted that three amino acids would be changed with each single mutation. Additionally work on human hemoglobin showed that mutations changed only one amino acid at a time. In the experimental work described in this paper, Crick induced deletion or insertion mutations in the rII gene of bacteriophage T4. Single-base insertions or deletions should produce a shift in the “reading frame” of the triplet code. His group was able to show that a single mutation gave a mutant phenotype, but a combination of a single base addition and a single base deletion near to one another produced a normal phenotype. They also later showed that 2-base deletions or insertions conferred mutant phenotypes but that 3-base insertions or deletions were almost always wildtype.
  • Translation, or protein synthesis, is directed in eukayotic cells by an mRNA molecule. Translation can be seen to occur in two phases: (1) information transfer, in which RNA base sequence of the mRNA determines the sequence of amino acids and (2) chemical processes, in which the peptide bonds between the adjacent amino acids are formed. The components required for translation include: mRNA, ribosomes, tRNA, aminoacyl tRNA synthetases, and accessory proteins involved in initiation, elongation and termination.
  • Ribosomes consist of two subunits that are named based on their size. Each of these subunits contains ribosomal RNA and protein. Ribosome subunit size is determined by their sedimentation rate an is indicated by S (Svedberg unit). In bacteria the large ribosome subunit is 50S, and the small is 30S. In eukaryotes the large subunit is 60S, and the small is 40S. The events translation initiation are the binding of the small ribosomal subunit to the mRNA molecule and the binding of the charged tRNA that bears the first amino acid of the polypeptide chain. The binding of the small ribosomal subunit is mediated by the 16S RNA that is part of the small ribosomal subunit in prokaryotes. In eukaryotes the 5´ methyl cap of the mRNA is involved in binding the small ribosomal subunit. The small ribosomal subunit, mRNA and tRNA complex recruits the large ribosomal subunit.
  • Elongation can be thought to involve three processes: 1) aligning each aminoacylated tRNA, 2) forming the peptide bond to add the new amino acid to the polypeptide chain, and 3)moving the ribosome along the mRNA by three more bases (one codon). EF-Tu (prokaryotes) or EF-1alpha (eukaryotes) aligns each new tRNA into the A site of the large subunit. This process requires the hydrolysis of GTP. When the A site is filled, a peptidyl transferase activity catalyzes the formation of the peptide bond between the amino acid in the A site and the adjacent amino acid in the P site. The formation of the peptide bond requires several proteins and RNA molecules of the large subunit and some evidence suggests that the peptide bond formation is catalyzed by an RNA molecule. After the peptide bond is formed the ribosome translocates 3 bases, the tRNA from the P-site that is no longer attached to its amino acid moves to the E site, and the tRNA with amino acid from the A site is now occupies the P site. The A site is now empty. This is known as the “posttranslocation state” for the ribosome. A charged tRNA bearing the next amino acid in the polypeptide chain binds to the codon of the mRNA and occupies the A site. The uncharged tRNA in the E site is released. This is known as the posttranslocation state. This process is repeated, with the ribosome reading the mRNA molecule one codon at a time.
  • Elongation proceeds until a stop codon is reached. There are three stop codons in the genetic code: UAG, UGA, UAA. There is no tRNA that bears an anticodon that can read any of the stop codons (the anticodons would be AUC, ACU, or AUU). Therefore no tRNA can fill the empty A site of the elongdation complex. Instead the A site is filled by a release factor, and the amino acid is cleaved from the tRNA that occupies the P site of the ribosome.

Transcription and Translation Transcription and Translation Presentation Transcript

  • Transcription and Translation The Relationship Between Genes and Proteins
  • Table of Contents
    • History: linking genes and proteins
    • Getting from gene to protein: transcription
      • Evidence for mRNA
      • Overview of transcription
      • RNA polymerase
      • Stages of Transcription
        • Promoter recognition
        • Chain initiation
        • Chain elongation
        • Chain termination
      • mRNA Synthesis/Processing
      • References
  • Table of Contents (continued)
    • Getting from gene to protein: genetic code
    • Getting from gene to protein: translation
      • Translation Initiation
      • Translation Elongation
      • Translation Termination
      • References
  • History: linking genes and proteins
    • 1900’s Archibald Garrod
      • Inborn errors of metabolism: inherited human metabolic diseases ( more information )
        • Genes are the inherited factors
        • Enzymes are the biological molecules that drive metabolic reactions
        • Enzymes are proteins
        • Question:
        • How do the inherited factors, the genes, control the structure and activity of enzymes (proteins)?
  • History: linking genes and proteins
    • Beadle and Tatum (1941) PNAS USA 27, 499–506.
    • Hypothesis:
      • If genes control structure and activity of metabolic enzymes, then mutations in genes should disrupt production of required nutrients, and that disruption should be heritable.
    • Method:
      • Isolated ~2,000 strains from single irradiate spores ( Neurospora ) that grew on rich but not minimal medium. Examples: defects in B1, B6 synthesis.
    • Conclusion:
      • Genes govern the ability to synthesize amino acids, purines and vitamins.
  • History: linking genes and proteins
    • 1950s: sickle-cell anemia
      • Glu to Val change in hemoglobin
      • Sequence of nucleotides in gene determines sequence of amino acids in protein
      • Single amino acid change can alter the function of the protein
    • Tryptophan synthase gene in E. coli
      • Mutations resulted in single amino acid change
      • Order of mutations in gene same as order of affected amino acids
  • From gene to protein: transcription
    • Gene sequence (DNA) recopied or transcribed to RNA sequence
    • Product of transcription is a messenger molecule that delivers the genetic instructions to the protein synthesis machinery: messenger RNA (mRNA)
  • Transcription: evidence for mRNA
    • Brenner, S., Jacob, F. and Meselson, M. (1961) Nature 190 , 576–81.
    • Question: How do genes work?
      • Does each one encode a different type of ribosome which in turn synthesizes a different protein, OR
      • Are all ribosomes alike, receiving the genetic information to create each different protein via some kind of messenger molecule?
  • Transcription: evidence for mRNA
    • E. coli cells switch from making bacterial proteins to phage proteins when infected with bacteriophage T4.
    • Grow bacteria on medium containing “heavy” nitrogen ( 15 N) and carbon ( 13 C).
    • Infect with phage T4.
    • Immediately transfer to “light” medium containing radioactive uracil.
  • Transcription: evidence for mRNA
    • If genes encode different ribosomes, the newly synthesized phage ribosomes will be “light”.
    • If genes direct new RNA synthesis, the RNA will contain radiolabeled uracil.
    • Results:
      • Ribosomes from phage-infected cells were “heavy”, banding at the same density on a CsCl gradient as the original ribosomes.
      • Newly synthesized RNA was associated with the heavy ribosomes.
      • New RNA hybridized with viral ssDNA, not bacterial ssDNA.
  • Transcription: evidence for mRNA
    • Conclusion
      • Expression of phage DNA results in new phage-specific RNA molecules (mRNA)
      • These mRNA molecules are temporarily associated with ribosomes
      • Ribosomes do not themselves contain the genetic directions for assembling individual proteins
  • Transcription: overview
    • Transcription requires:
    • ribonucleoside 5´ triphosphates:
      • ATP, GTP, CTP and UTP
      • bases are adenine, guanine, cytosine and uracil
      • sugar is ribose (not deoxyribose)
    • DNA-dependent RNA polymerase
    • Template (sense) DNA strand
    • Animation of transcription
  • Transcription: overview
    • Features of transcription:
    • RNA polymerase catalyzes sugar-phosphate bond between 3´-OH of ribose and the 5´-PO 4 .
    • Order of bases in DNA template strand determines order of bases in transcript.
    • Nucleotides are added to the 3´-OH of the growing chain.
    • RNA synthesis does not require a primer.
  • Transcription: overview
    • In prokaryotes transcription and translation are coupled. Proteins are synthesized directly from the primary transcript as it is made.
    • In eukaryotes transcription and translation are separated. Transcription occurs in the nucleus, and translation occurs in the cytoplasm on ribosomes.
    • Figure comparing eukaryotic and prokaryotic transcription and translation.
  • Transcription: RNA Polymerase
    • DNA-dependent
      • DNA template, ribonucleoside 5´ triphosphates, and Mg 2+
    • Synthesizes RNA in 5´ to 3´ direction
    • E. coli RNA polymerase consists of 5 subunits
    • Eukaryotes have three RNA polymerases
      • RNA polymerase II is responsible for transcription of protein-coding genes and some snRNA molecules
      • RNA polymerase II has 12 subunits
      • Requires accessory proteins (transcription factors)
      • Does not require a primer
  • Stages of Transcription
    • Promoter Recognition
    • Chain Initiation
    • Chain Elongation
    • Chain Termination
  • Transcription: promoter recognition
    • Transcription factors bind to promoter sequences and recruit RNA polymerase .
    • DNA is bound first in a closed complex. Then, RNA polymerase denatures a 12–15 bp segment of the DNA (open complex).
    • The site where the first base is incorporated into the transcription is numbered “+1” and is called the transcription start site.
    • Transcription factors that are required at every promoter site for RNA polymerase interaction are called basal transcription factors.
  • Promoter recognition: promoter sequences
    • Promoter sequences vary considerably.
    • RNA polymerase binds to different promoters with different strengths; binding strength relates to the level of gene expression
    • There are some common consensus sequences for promoters:
      • Example: E. coli –35 sequence (found 35 bases 5´ to the start of transcription)
      • Example: E. coli TATA box (found 10 bases 5´ to the start of transcription)
  • Promoter recognition: enhancers
    • Eukaryotic genes may also have enhancers.
    • Enhancers can be located at great distances from the gene they regulate, either 5´ or 3´ of the transcription start, in introns or even on the noncoding strand.
    • One of the most common ways to identify promoters and enhancers is to use a reporter gene.
  • Promoter recognition: other players
    • Many proteins can regulate gene expression by modulating the strength of interaction between the promoter and RNA polymerase.
    • Some proteins can activate transcription (upregulate gene expression).
    • Some proteins can inhibit transcription by blocking polymerase activity.
    • Some proteins can act both as repressors and activators of transcription.
  • Transcription: chain initiation
    • Chain initiation :
    • RNA polymerase locally denatures the DNA.
    • The first base of the new RNA strand is placed complementary to the +1 site.
    • RNA polymerase does not require a primer.
    • The first 8 or 9 bases of the transcript are linked. Transcription factors are released, and the polymerase leaves the promoter region.
    • Figure of bacterial transcription initiation .
  • Transcription: chain elongation
    • Chain elongation :
    • RNA polymerase moves along the transcribed or template DNA strand.
    • The new RNA molecule (primary transcript) forms a short RNA-DNA hybrid molecule with the DNA template.
  • Transcription: chain termination
    • Most known about bacterial chain termination
    • Termination is signaled by a sequence that can form a hairpin loop.
    • The polymerase and the new RNA molecule are released upon formation of the loop.
    • Review the transcription animation.
  • Transcription: mRNA synthesis/processing
    • Prokaryotes: mRNA transcribed directly from DNA template and used immediately in protein synthesis
    • Eukaryotes: primary transcript must be processed to produce the mRNA
      • Noncoding sequences (introns) are removed
      • Coding sequences (exons) spliced together
      • 5´-methylguanosine cap added
      • 3´-polyadenosine tail added
  • Transcription: mRNA synthesis/processing
    • Removal of introns and splicing of exons can occur several ways
      • For introns within a nuclear transcript, a spliceosome is required.
        • Splicesomes protein and small nuclear RNA (snRNA)
        • Specificity of splicing comes from the snRNA, some of which contain sequences complementary to the splice junctions between introns and exons
      • Alternative splicing can produce different forms of a protein from the same gene
      • Mutations at the splice sites can cause disease
        • Thalassemia • Breast cancer (BRCA 1)
  • Transcription: mRNA synthesis/processing
    • RNA splicing inside the nucleus on particles called spliceosomes.
    • Splicesomes are composed of proteins and small RNA molecules (100–200 bp; snRNA).
    • Both proteins and RNA are required, but some suggesting that RNA can catalyze the splicing reaction.
    • Self-splicing in Tetrahymena : the RNA catalyzes its own splicing
    • Catalytic RNA: ribozymes
  • From gene to protein: genetic code
    • Central Dogma
      • Information travels from DNA to RNA to Protein
        • Is there a one-to-one correspondence between DNA, RNA and Protein?
          • DNA and RNA each have four nucleotides that can form them; so yes, there is a one-to-one correspondence between DNA and RNA.
          • Proteins can be composed of a potential 20 amino acids; only four RNA nucleotides: no one-to-one correspondence.
          • How then does RNA direct the order and number of amino acids in a protein?
  • From gene to protein: genetic code
    • How many bases are required for each amino acid?
        • (4 bases) 2bases/aa = 16 amino acids—not enough
        • (4 bases) 3bases/aa = 64 amino acid possibilities
    • Minimum of 3 bases/aa required
    • What is the nature of the code?
      • Does it have punctuation? Is it overlapping?
      • Crick, F.H. et al . (1961) Nature 192 , 1227–32. ( http://profiles.nlm.nih.gov/SC/B/C/B/J/ )
      • 3-base, nonoverlapping code that is read from a fixed point.
  • From gene to protein: genetic code
    • Nirenberg and Matthaei: in vitro protein translation
      • Found that adding rRNA prolonged cell-free protein synthesis
      • Adding artificial RNA synthesized by polynucleotide phosphorylase (no template, UUUUUUUUU) stimulated protein synthesis more
      • The protein that came out of this reaction was polyphenylalanine (UUU = Phe)
      • Other artificial RNAs: AAA = Lys; CCC =Pro
  • From gene to protein: genetic code
    • Nirenberg:
      • Triplet binding assay: add triplet RNA, ribosomes, binding factors, GTP, and radiolabeled charged tRNA ( figure )
        • UUU trinucleotide binds to Phe-tRNA
        • UGU trinucleotide binds to CYS-tRNA
      • By fits and starts the triplet genetic code was worked out.
      • Each three-letter “word” (codon) specifies an amino acid or directions to stop translation.
      • The code is redundant or degenerate: more than one way to encode an amino acid
  • From gene to protein: Translation
    • Components required for translation:
      • mRNA
      • Ribosomes
      • tRNA
      • Aminoacyl tRNA synthetases
      • Initiation, elongation and termination factors
    • Animation of translation
  • Translation: initiation
    • Ribosome small subunit binds to mRNA
    • Charged tRNA anticodon forms base pairs with the mRNA codon
    • Small subunit interacts with initiation factors and special initiator tRNA that is charged with methionine
    • mRNA-small subunit-tRNA complex recruits the large subunit
    • Eukaryotic and prokaryotic initiation differ slightly
  • Translation: initiation
    • The large subunit of the ribosome contains three binding sites
      • Amino acyl (A site)
      • Peptidyl (P site)
      • Exit (E site)
    • At initiation,
      • The tRNA fMet occupies the P site
      • A second, charged tRNA complementary to the next codon binds the A site.
  • Translation: elongation
    • Elongation
    • Ribosome translocates by three bases after peptide bond formed
    • New charged tRNA aligns in the A site
    • Peptide bond between amino acids in A and P sites is formed
    • Ribosome translocates by three more bases
    • The uncharged tRNA in the A site is moved to the E site.
  • Translation: elongation
      • EF-Tu recruits charged tRNA to A site. Requires hydrolysis of GTP
      • Peptidyl transferase catalyzes peptide bond formation (bond between aa and tRNA in the P site converted to peptide bond between the two amino acids)
      • Peptide bond formation requires RNA and may be a ribozyme-catalyzed reaction
  • Translation: termination
    • Termination
    • Elongation proceeds until STOP codon reached
      • UAA, UAG, UGA
    • No tRNA normally exists that can form base pairing with a STOP codon; recognized by a release factor
    • tRNA charged with last amino acid will remain at P site
    • Release factors cleave the amino acid from the tRNA
    • Ribosome subunits dissociate from each other
    • Review the animation of translation