I.    Transcription
    A.    Chemistry of the reaction to make an RNA polymer
        1.    Use NTPs as energy source. Remove Pyrophosphate.
        2.    Attach remaining monophosphate in a 5'-3' linkage, similar to DNA linkages.
    B.    RNA Polymerases
        1.    Do not need a preexisting 3'OH. Can attach two nucleotides together to start an RNA
                polymer.
        2.    Types
            a.    RNA pol I: transcribes the large rRNAs in eukaryotes
            b.    RNA pol II: transcribes the primary RNA transcript that
                   will become mRNA in eukaryotes
            c.    RNA pol III: transcribes small RNAs in eukaryotes (tRNAs, snRNAs, etc.)
    C.    Regulatory Sequences
        1.    Promoter
            a.    Loads RNA polymerase II onto the DNA where it can then find the startpoint of
                   transcription.
            b.    Located approximately 30-50 nucleotides upstream of the +1 startpoint.
            c.    Most common type is the TATA box.
                -Consists of a consensus sequence TATAA/TA that is the site of assembly of the
                  general transcription factors.
            d.    Alternative promoter types exist but are not included on the exam.
        2.    Upstream Promoter Elements
            a.    Located approximately 200-500 basepairs upstream from +1 startpoint.
            b.    Many different types have been identified.
            c.    Bind the specific transcription factors.
            d.    Believed to modify the ability of the general transcription factors to load on RNA
                       pol II.
            e.    Bind both activators and/or repressors.
                    -activators can be histone acetylases, acetylating histones at their amino termini.
                    -activators can directly bind other cofactors and make it easier for the general
                      transcription to bind at the TATA box.
                    -repressors can compete with activators for binding sites or suppress their activation
                      domains by binding to them.
                    -repressors can directly bind to the general transcription factors and inhibit further
                     assembly.
        3.    Enhancers (Silencers)
            a.    Unusual sequences that can modify transcription rates at large distances from the +1
                   startpoint (1-5kb) and on either side of the gene and in either orientation.
            b.    Bind additional specific transcription factors that can either upregulate or downregulate.
            c.    Believed to interact with the general transcription factors by looping the DNA
                   to bring the enhancer(silencer) binding proteins to the vicinity of the promoter.
        4.    How RNA polymerase gets loaded on:
            a.    TFIID consists of TBP (TATA binding protein) and a complex of tafs.
            b.    TFIID complex binds to the TATA box through the TBP.
            c.    Inhibitors of TBP can bind to it and stop the assembly process.
            d.    If no inhibitor binds TPB, the remainder of the general transcriptions assemble.
            e.    This includes TFIIF bound to RNA polymerase II.
            f.    Once all are assembled, TFIIH acts.
                -Its protein kinase activity attaches phosphates to RNA polymerase II at its CTD
                -Its helicase activity melts the hydrogen bonds of the DNA to expose the template.
            g.    RNA polymerase II will begin transcription and clear away from the promoter region.
      D.    Many eukaryotic genes are regulated at the level of transcription initiation.
            1..    Example: experiment where liver mRNAs were extracted from the cytoplasm of
                   cells and shown to hybridize positively to cDNA probes from known liver protein genes
                    Other cell types known not to make these proteins did not show positive hybridization
                    to these probes.
      E.    Antitermination
            1.    RNA polymerase II often pauses during elongation of transcription and will only
                    complete transcription when signalled to do so.
            2.    One example is the tat protein of HIV.
                    - Tat binds to a sequence near the 5' end of the transcript called TAR, part of a stem-
                    loop structure.
                    -Cellular factors bind to another stem-loop structure.
                    -Together they coordinate interaction with other cellular proteins that includes
                      cdk9, a protein kinase that phosphorylates the CTD of the stalled RNA pol II and
                      causes it to continue transcription.
            3.    Another example is the control of the hsp70 transcript which responds to stress in cells.
                    -The non-stressed cell begin transcription but the RNA polymerase II pauses.
                    -In response to stress, a protein called HSTF changes shape from an inactive to an
                      active form.
                    -It now can bind near promoter-proximal elements called GAGA.
                    -In some way this causes the stalled RNA polymerase II to resume transcription as
                     well as stimulating more rounds of transcription by other RNA polymerase IIs.
    F.    Processing the primary RNA transcript
        1.    Capping the 5' end.
             a.    The CAP is a G nucleotide methylated at the N7 position of the base and
                    attached to the first nucleotide of the RNA transcript by a 5'-5' triphosphate
                    bridge. In vertebrates, the 2'O of the ribose is methylated in the first and second
                    nucleotides also.
            b.    This CAP is used during the initiation of translation to help set the reading frame.
        2.       3'-end processing (cleavage and polyadenylation)
            a.   In the primary transcript, a poly-adenylation signal is made that reads AAUAA.
            b.   The actual cleavage and poly-A addition site is about 20-30 nucleotides downstream.
            c.    RNA pol II continues transcription past these sites and makes a G/U rich sequence in
                    the transcript. It continues transcribing.
            d.   CPSF binds to the AAUAA signal.
            e.   This facilitates the binding of the cleavage factors (CFI and CFII)and CstF, which binds
               to the G/U-rich sequence.
            f.   This allows PAP (poly-A polymerase) to bind.
            g.   The cleavage factors cleave the transcript at the poly-A site and PAP attaches the
                  poly-A tail.
            h.  At first the poly-A addition is slow. Then the CFI, CFII, and CStF release.
            i.  PABII binds to the poly-A tail and rapid polyadenylation proceeds. About 200 A
               residues get attached. The rest of the transcript beyond the cleavage site is degraded.
        3.   Removal of introns
            a.   Eukaryotic genes contain intervening sequences called introns.
            b.   RNA polymerase II transcribes these introns.
            c.   They must be removed from the transcript and the exons  (what is left) connected into a
               continuous mRNA sequence.
            d.   This is done by a mechanism known as splicing.
            e.  Evidence for splicing comes from a technique known as R-looping
                -mRNA is hybridized to single-stranded DNA strands (hydrogen bonds have
                  been melted).
                -Complementary sequences will hybridize.
                -Intron sequences will have no complementary sequence in the mRNA.
                -They "loop out" and can be seen in electron micrographs.
            f.    The splicing signal in the primary transcript has three major features.
                -There is a 100% conserved GU at the 5'end of the intron.
                -There is a 100% conserved AG at the 3' end of the intron.
                -There is an A nucleotide about 20-30- nucleotides from the 3'end of the intron that
                  is called the branch point and is 100% conserved.
            g.    The intron is removed by two transesterification reactions.
                -First, the 2'OH on the ribose of the branch point A attacks the 3' end of exon 1.
                Tthis breaks the phosphodiester bond between the last nucleotide of exon 1 and the
                 first nucleotide of the intron.
                -A bond forms between the 5'P of the first intron nucleotide and the 2'OH of the A.
                -Then, the 3' end of exon 1 (the 3'OH) attacks the 5' end of exon 2.
                -This breaks the bond between the 3' end of the intron and the 5'end of exon 2.
                -The 3' end of exon 1 links to the 5'end of exon 2 by a phosphodiester linkage.
                -The intron (in lariat form) is removed and degrades.
                -See page 417 in the text for diagrams of this reaction.
            h.    These reactions are catalyzed and controlled by nuclear particles called snurps.
                -consist of small nuclear RNAs complexed to proteins.
                -several kinds are involved, called U1, U2, etc.
                -U1 has snRNA that is complementary to the GU and flanking sequences at the exon/
                 intron junction. It binds first during the splicing reaction
                -U2 has snRNA complementary to the consensus sequence surrounding the A
                 branchpoint but not the A itself which bulges out. This facilitates the first reaction.
                -After U2 binds, the remaining snurps bind forming the spliceosome.
                -The RNA-RNA interactions between the transcript and the snurps and between the
                 snurps rearrange as the reactions continue, facilitating the process.
                -Finally, the spliceosomes, still attached to the intron, leave at the end of the splicing
                 reactions and the snurps disassemble.
    G.    Control of Gene Expression at the level of 3' end processing
         1.       Choice of cleavage and polyadenylation site can change subsequent patterns of
                of splicing and/or translation, making different proteins or forms of a protein.
          2.    Our example was the antibody molecule IgM that can exist as either a membrane-
                bound or secreted molecule, depending on the differentiation state of the B cell.
                -Earlier in the differentiation pathway, the B cell makes the membrane-bound molecule.
                -This requires that the COOH terminal contain two exons that code for a hydrophobic
                  region that can associate with the cell membrane.
                -To do this, an early poly-A signal is ignored and a later one recognized on the primary
                   RNA transcript.
                -Cleavage and polyadenylation using this second signal gives rise to a mRNA that
                 contains the necessary exons and is translated into the membrane bound protein.
                -Later, when the cell is ready to secrete the antibody, it is signalled to recognize the
                   the first poly-A signal and uses it.
                -Following cleavage and poly-adenylation, the subsequent splicing events make a
                mRNA that does not contain the exons for the hydrophobic region and the protein
                made will not associate with the cell membrane but will be secreted.
     H. Control at the level of splicing.
        1.    sex-determination in the developing fruit-fly
            a.   Early in embryogenesis, an early promoter of the sex-lethal gene is active in female
                embryos but not in males. Early sex-lethal is made.
            b.    Later, the late promoter is used by both sexes.
            c.    The female sex-lethal transcript is spliced differently because the early sex-lethal protein,
                    an RNA binding protein causes removal of an exon containing a stop codon with an
                    intron allowing a functional late sex-lethal protein to be made. In males no alternate
                    splicing occurs, stop codon remains, so no protein.
            d.    Late sex-lethal protein autoregulates its own production by a similar mechanism.
            e.    Late sex-lethal protein also causes alternative splicing of the transformer (tra) gene
                    in females, similarly. No such regulation in males.
            f.    Functional tra protein made in females, not in males.
            g.    tra binds to tra-2 (another RNA binding protein)
            h.    tra/tra2 upregulates the splicing of an intron usually ignored but now spliced in females.
            i.    Since no tra made in males, no tra/tra2, so no upregulation and therefore no alternative
                splicing.
            j.    h. and i. are happening on a gene called double-sex, producing different forms of the
                  of the mRNA and therefore of the protein.
            k.    double-sex female activates transcription of genes responsible for female sexual
                    differentiation.
            l.    double-sex male activates transcription of genes responsible for male sexual
                    differentiation.
    I.  Control at the level of mRNA stability
        1.    The 3' untranslated region of eukaryotic mRNAs can contain repeated sequences of
              AUUUA or similar sequences that signal the mRNA to be degraded by nucleases.
            a.    This causes these mRNAs to have very short half-lives
            b.    The proteins coded for by these mRNAs are usually central to growth regulation for
                    the cell.
            c.    If a long-lived mRNA like b-globin has its 3'UTR replaced by the AUUUA sequences
                    from a short-lived mRNA, it becomes short-lived also.
            d.    Histones are regulated in a cell-cycle manner.
                    -Their mRNAs are unstable except during S phase when they become stable.
                    -due to stem-loop in the 3'UTR.
        2.    Example we looked at was the mRNA for the transferin receptor.
            a.    Has several AUUUA-like repeats in its 3'UTR.
            b.    Allows the formation of stem-loops that contain an iron-response element (IRE).
            c.    An IRE binding protein exists in two possible conformations.
                   -when iron amounts are low in the cell, it is active and binds to the IREs.
                   -This protects the mRNA from degredation.
                   -Therefore, translation will occur and more transferin receptor will be made.
                   -This will allow more iron to be brought into the cell.

II.    Amino Acid and Protein Structure
    A.    Amino Acid Structure
        1.   alpha-carbon bonded to H, NH2, COOH and a side chain, R
        2.    R determines the classification of amino acids, based on chemical characteristics
            a.    hydrophobic
            b.    polar, but not charged
            c.    basic, + charged
            d.    aidic, - charged
        3.    exists in 2 possible stereoisomeric forms, D and L
            a.    only L used in cells.
    B.    Primary Structure of Polypeptides
        1. The peptide bond
           a.    resonance and planarity
            b.    formed by condensation reactions between the COOH of one amino acid
                    and the NH2 of the next
            c.    Free NH2 end is called the amino terminus; free COOH end is the carboxy terminus
            d.    Sequence of amino acids is called the primary structure.
    C.    Secondary Structure
        1.    alpha-helix
            a.    stabilized by H-bonds between one amino acid peptide linkage and the peptide
                    linkage 4 residues away.
            b.    R chains project to the outside of the helix.
        2.    beta-pleated sheet
            a.    linear arrays of amino acids stabilized by hydrogen bonding between adjacent amino
                    acids
            b.    R chains project above or below the pleated sheet
            c.    can run in parallel or in antiparallel arrays
        3.    motifs of secondary structure can be recognized in peptide chains.
    D.    Tertiary Structure
        1.    considers all possible interactions of the side-chains of the amino acids
        2.    stabilized by hydrogen bonds, ionic interactions, hydrophobic interaction, and
                van der Waal's forces.
        3.    disulfide linkages between cysteines can also influence the tertiary structure
        4.    creates structural domains
        5.    can also characterize functional domains that participate in particular protein functions.
    E.    Quarternary Structure
        1.    interacting subunits needed for a protein to function. (same or different)
        2.    hemoglobin example