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  1. DNA Transcription or RNA Synthesis
    1. Transcription-the synthesis of RNA under the direction of DNA
      1. The RNA product is complementary to the DNA template
      2. An adenine nucleotide in the DNA template directs the incorporation of a uracil nucleotide in the RNA; otherwise, the base pair rules are the same as for DNA replication
      3. Three types of RNA are produced by transcription
        1. mRNA carries the message that directs the synthesis of proteins
        2. tRNA carries amino acids during protein synthesis
        3. rRNA molecules are components of the ribosomes
    2. Transcription in procaryotes
      1. Procaryotic mRNA can code for one polypeptide (monogenic) or many polypeptides (polygenic); in addition to coding regions, mRNA molecules may have untranslated regions
        1. Leader sequences consist of 25 to 150 bases at the 5¢ end of the mRNA, and precede the initiation codon
        2. Spacer regions separate the segments that code for individual polypeptides in polygenic mRNAs
        3. Trailer regions are found at the 3¢ end of the mRNA after the last termination codon
      2. RNA polymerase (a large multi-subunit enzyme) is responsible for the synthesis of RNA
        1. The core enzyme (a2,b,b' subunits) catalyzes RNA synthesis
        2. The sigma subunit (sigma factor) is not catalytic, but helps the core enzyme bind DNA at the appropriate site
      3. A promoter is the region of the DNA to which RNA polymerase binds in order to initiate transcription; sequences centered at 35 and 10 base pairs before the transcription starting point are important in directing RNA polymerase to the promoter
      4. Terminators are regions of the DNA that signal termination of the transcription process
    3. Transcription in eucaryotes
      1. There are three major RNA polymerases
        1. RNA polymerase II-catalyzes mRNA synthesis; it requires several initiation factors and recognizes promoters that have several important elements (rather than just two as seen in procaryotes)
        2. RNA polymerase I-catalyzes rRNA (5.8S, 18S, and 28S) synthesis
        3. RNA polymerase III-catalyzes tRNA and 5S rRNA synthesis
      2. Transcription yields large monogenic RNA precursors (heterogeneous nuclear RNA; hnRNA) that must be processed by posttranscriptional modification to produce mRNA
        1. Adenylic acid is added to the 3¢ end to produce a polyA sequence about 200 nucleotides long (polyA tail)
        2. 7-methylguanosine is added to the 5¢ end by a tri-phosphate linkage (5' cap)
        3. These two modifications are believed to protect the mRNA from exonuclease digestion
      3. Eucaryotic genes are split or interrupted such that the expressed sequences (exons) are separated from one another by intervening sequences (introns); the introns are represented in the primary transcript but are subsequently removed by a process called RNA splicing; some splicing is self-catalyzed by RNA molecules called ribozymes; interrupted genes have been found in cyanobacteria and archaea, but not in other procaryotes
  2. Protein Synthesis
    1. Translation-the synthesis of a polypeptide chain directed by the nucleotide sequence in a mRNA molecule
    2. Ribosome-site of translation 2. Polyribosome-complex of mRNA with several ribosomes

    3. Transfer RNA and amino acid activation
      1. The first stage of protein synthesis is the attachment of amino acids to tRNA molecules (catalyzed by aminoacyl-tRNA synthetase); this process is referred to as amino acid activation
      2. Each tRNA has an acceptor end and can only carry a specific amino acid; it also has an anticodon triplet that is complementary to the mRNA codon triplet
    4. The ribosome-complex organelle constructed from several rRNA molecules and many polypeptides; has two subunits (in procaryotes: 50S and 30S)
    5. Initiation of protein synthesis
      1. fMet-tRNA (in bacteria, Met-tRNA in archaea and eucaryotes) binds the small subunit of the ribosome; they bind the mRNA at a special initiator codon (AUG); then the large subunit of the ribosome binds
      2. Three protein initiation factors are also required in procaryotes (eucaryotes require more initiation factors)
    6. Elongation of the polypeptide chain
      1. Elongation involves the sequential addition of amino acids to the growing polypeptide chain; several polypeptide elongation factors are required for this process
      2. The ribosome has three sites for binding tRNA molecules: peptidyl site (P site), aminoacyl site (A site), and exit site (E site)
      3. Each new amino acid is positioned in the A site by its tRNA, which has an anticodon that is complementary to the codon on the mRNA molecule
      4. The ribosomal enzyme peptidyl transferase catalyzes the formation of the peptide bonds between adjacent amino acids; the 23S rRNA is a major component of this enzyme
      5. After each amino acid is added to the chain, translocation occurs and thereby moves the ribosome to position the next codon in the A site
    7. Termination of protein synthesis
      1. Takes place at any one of three special codons (UAA, UAG, or UGA)
      2. Three polypeptide release factors aid in the recognition of these codons
      3. The ribosome hydrolyzes the bond between the completed protein and the final tRNA, and the protein is released from the ribosome, which then dissociates into its two component subunits
    8. Protein synthesis is expensive, using five high-energy bonds to add one amino acid to the chain
    9. Protein folding and molecular chaperones
      1. Molecular chaperones-special helper proteins that aid the nascent polypeptide in folding to its proper shape; many have been identified and they include heat shock proteins and stress proteins; in addition to helping polypeptides fold, chaperones are important in the transport or protein across membranes
      2. Protein conformation is a direct function of amino acid sequence; proteins have self-folding, structurally independent regions called domains
    10. Protein splicing-before folding, part of the polypeptide is removed; such splicing removes intervening sequences (inteins) from the sequences (exteins) that remain in the final product
  3. Regulation of mRNA Synthesis
    1. Regulation of mRNA synthesis (and thereby enzyme synthesis) provides a long-term regulatory mechanism that very effectively conserves energy and raw material and maintains overall balance of cell proteins in response to major changes in environmental conditions; it complements but is less rapid than regulation of enzyme activity
    2. Induction and repression
      1. Synthesis of enzymes involved in catabolic pathways can be inducible, and the initial substrate of the pathway (or some derivative of it) is usually the inducer; induction increases the amount of mRNA encoding the enzymes
      2. Synthesis of enzymes involved in anabolic pathways is repressible and the end product of the pathway usually acts as a corepressor; repression decreases the amount of mRNA encoding the enzymes
    3. Negative control
      1. The rate of mRNA synthesis is controlled by repressor proteins, which bind to specific sites on the DNA called operators
      2. In inducible systems, the repressor protein is active until bound to the inducer (binding of inducer inactivates the repressor) whereas in repressible systems, the repressor is inactive until bound to the corepressor (binding of corepressor activates the repressor)
      3. In bacteria, a set of structural genes controlled by a particular operator and promoter is called an operon
      4. The lactose operon is an excellent example of negative regulation; binding of the lac repressor to the lac operator may either inhibit RNA polymerase binding or block the movement of RNA polymerase
    4. Positive control
      1. Positive control occurs when an operon can function only in the presence of a controlling factor
      2. The lactose operon is regulated by catabolite activator protein (CAP); the activity of CAP is regulated by cAMP; cAMP activates CAP so that it binds a specific site on the DNA, stimulating transcription
  4. Attentuation
    1. There are two decision points for regulating transcription of anabolic pathways: initiation and continuation of transcription; attenuation regulates continuation of transcription
    2. In systems where transcription and translation are tightly coupled, ribosome behavior in the leader region of the mRNA can control continuation of transcription
      1. If ribosomes actively translate the leader region (attenuator), which contains several codons for the amino acid product of the operon, a transcription terminator forms and transcription will not continue
      2. If ribosomes stall during translation of the leader region because the appropriate charged aminoacyl-tRNA is absent, the terminator does not form and transcription will continue
  5. Global Regulatory Systems
    1. Overview
      1. Global regulatory systems-systems that affect many genes and pathways simultaneously, allowing for both independent regulation of operons as well as cooperation of operons
      2. Global regulation can be accomplished by several mechanisms, including the use of a single regulator protein (repressor or activator) to regulate several operons, use of different sigma factors, and the use of nonprotein regulators
      3. Regulon-a collection of genes or operons controlled by a common regulatory protein
    2. Catabolite repression
      1. Diauxic growth-a biphasic growth pattern observed when a bacterium is grown on two different sugars (e.g., glucose and lactose)
      2. For E. coli, availability of glucose (the preferred carbon and energy source) causes a drop in cAMP levels, resulting in the deactivation of CAP (a positive regulator of several catabolic pathways, including the lactose operon); deactivation of CAP allows the bacterium to use glucose preferentially over another sugar when both are present in the environment
    3. Regulation by sigma factors and control of sporulation
      1. Bacteria produce a number of different sigma factors; each enables RNA polymerase to recognize and bind to specific promoters
      2. Alteration of the sigma factors available to RNA polymerase changes gene expression
      3. This has been demonstrated in a number of systems including heat shock response in E. coli and sporulation in B. subtilis
    4. Regulation by antisense RNA and the control of porin proteins-antisense RNA is complementary to an RNA molecule and specifically binds to it, thereby blocking DNA replication, mRNA synthesis, or translation, depending on the nature of the RNA target
  6. Two-Component Phosphorelay Systems
    1. A signal transduction system that uses transfer of phosphoryl groups to control gene transcription and protein activity
    2. In the sporulation regulation system, sequential transfer of a phosphoryl group from a sensor kinase to a transcription regulator, via two other proteins, allows the bacterium to respond to environmental conditions
    3. Chemotaxis-in this system, attractants (or repellants) are detected by chemotactic proteins, leading to transfer of phsophoryl groups to proteins that regulate the direction of rotation of the bacterial flagellum
  7. Control of the Cell Cycle
    1. The complete sequence of events extending from the formation of a new cell through the next division is called the cell cycle: it requires tight coordination of DNA replication and cell division
    2. There appear to be two separate controls for the cell cycle, one sensitive to cell mass and the other responding to cell length
    3. In E. coli, regulation of DNA replication involves DnaA protein, which binds to the origin of replication to initiate replication
    4. The initiation of septation requires both termination of DNA replication and the attainment of threshold lengths; how this occurs in unknown
    5. In rapidly growing bacterial cultures, initiation of a round of DNA replication can begin before a round of replication is finished

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