Remember: For Spring, 2005,  DNA repair is on exam two also. It is reviewed in Review Sheet One.

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, etc.)
        3.    Contain three activities
            a.    unwinding activity (helicase): allows the ds DNA to become single-stranded.
            b.    polymerizing activity:    forms the RNA transcript.
            c.    rewinding activity:    displaces the RNA transcript (about 10-15 nucleotides long) from the template strand
                   and allows the ds DNA to reform.
    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, loosening
                      the chromatin structure.
                    -activators can directly bind other cofactors and make it easier for the general
                      transcription to bind at the TATA box.
                    -repressors can be histone deacetylases. Deaceylated histones are more compactly packed.
                    -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. Sometimes found in introns.
            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.    In-vitro studies indicate the following mechanism.
            b.    TBP (TATA binding protein) binds to the TATA.
            c.    The remainder of the general transcriptions assemble.
            d.    This includes TFIIF bound to RNA polymerase II.
            e.    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.
            f.    RNA polymerase II will begin transcription and clear away from the promoter region.
        5.    in-vivo, A large complex called mediator interacts with the TATA box region factors and forms a
                bridge between these factors and specific transcription factors, often bound to enhancer elements.
    D.    Many eukaryotic genes are regulated at the level of transcription initiation.
    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, participates in nuclear
                    export, and protects the growing transcript in the nucleus.
            c.    First, capping enzymes remove the gamma phosphate from the 5'nucleotide of the transcript.
                   Then, GTP has Pyrophosphate removed and the GMP is attached to the 5' nucleotide by the 5'
                   -5' triphosphate bridge. Then the 7N of the guanine is methylated by methylase enzymes and the 2'OH is
                   of the first nucleotide of the transcript is also methylated. In vertebrates, the second nucleotide is also
                   methylated in this manner. S-adenosyl methionine (SAM) is the source of the methyl groups.
        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 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.
            i.  PABII (a poly-A binding protein 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) from the same cell.
                -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.
            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.
               -SR proteins bind to ESE (exonic splicing enhancers) and interact with splicing factors
                within introns on either side. This helps identify the exons.
                -Alternative splicing is often used in cells to make different forms of a protein from the same transcript.
            i. Sex determination in Drosophila is an example.
                -Early promoter of sxl gene used in females, not males. Makes early form of sxl.
                -Later promoter of sxl used in both males and females. Early sxl in females binds to a 3' splice junction
                   and prevents that intron being removed; the next 3' junction is used instead, removing everything, including
                    the exon. This does not happen in males, therefore the exon remains. It has a stop codon in-frame,
                    causing premature termination of translation and a non-functional sxl. Females make the functional
                    sxl.
                -The functional sxl in females regulates the splicing of the tra gene transcript in a similar fashion, causing
                   the removal of an exon that has a stop codon. Males do not remove this exon, therefore they make
                    non-functional tra protein.j
                -Female tra forms a complex with tra2 and Rbp 1. This works as an SR protein, binding the exon next to a  3'
                    splice junction, activating it. This causes inclusion of an exon and translation creates the female form of
                   double-sex protein. This represses male-specific gene transcription leading to the female phenotype. Males
                   are not able to enhance the splice junction, thus the exon is removed as part of a larger intron, and
                    translation forms the male double-sex  protein which represses female-specific genes and the male
                    phenotype is formed.
    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.    An example is the change from a membrane-bound form of antibody molecule in B cells before antigen
                activation (retains a hydrophobic region that interacts with the membrane) and the secreted form of the
                antibody, following activation (now has a hydrophilic region and no hydrophobic region). Due to
                the choice of a downstream poly-A signal used in the first case and an upstream signal used during
                the latter case. The resulting transcripts result in different splicing patterns and different sequences in the
                COOH terminal of the proteins.

II.    Genetic Code
    A.    Triplet code
    B.    Reading Frame
            1.    insertions or deletions cause frameshift mutations.
    C.    Amino acid codons worked out using the Nirenberg filter binding assay. (Understand this experiment).
            1.    Synthetic trinucleotides added to cell extract containing all amino acids, tRNAs, ribosomes, and cofactors
                    needed for translation.
            2. Divided into 20 tubes. One radioactive amino acid added to each tube.
            3.  Mixes filtered, trapping ribosomes and any attached trinucleotide and tRNA charged with amino acid on
                the filter.
            4. If filter is radioactive, indicates that the radioactive amino acid from that tube is that coded by the trinucleotide.

Begin here for exam 3, 2005

III.     Major Players in Translation

    A.    messenger RNA (eukaryotes)
        1.    Cap at 5' end
        2.    5' untranslated region
        3.    AUG start codon
        4.    open reading frame (coding region)
        5.    3' untranslated region
        6.    poly A tail

    B.    transfer RNA
        1.    stem-loop structures
        2.    anticodon-loop
            a.    codon-anticodon base pairing. Inosine often found in the anticodon.
            b.    wobble position allows incorrect base pairing between first anticodon base and third codon base
            c.    understand conncection between wobble and the degeneracy of the genetic code
        3.    amino acid attachment to 3'end.

    C.    amino-acyl-tRNA synthetase
        1.    catalyzes attachment of amino acid to either the 2' (class 1) or 3' (class 2) OH of the ribose of the
                adenine at the 3' end of tRNA molecule.
        2.    two step reaction
            a.    Pyrophosphate removed from ATP; AMP attached to amino acid through COOH
                    group.
            b.    AMP removed and amino acid attached to the tRNA.
        3.    Specificity of the enzyme assures the correct attachment.
        4.    20 groups of amino-acyl-tRNA synthetases, one group for each of the 20 amino acids.

    D.    Ribosomes
        1.    Two subunits, large and small
        2.    Consist of ribosomal RNAs and ribosomal proteins.
        3.    Contain the enzymatic activity needed during protein synthesis.
        4.    Function in the cytoplasm.

IV.    The Translation Mechanism
    A.    Initiation
        1.    Sets the reading frame. Small subunit binds eIF6 (keeps it from binding large subunit).
        2.    Small subunit, initiator tRNA, eIF2- GTP,  interact.
        3.    Other initiation factors (eIF4E) bind to the Cap on mRNA.
                Poly-A binding proteins interact with eIF4G, looping the 3' end to the 5' end of the mRNA.
        4.    complex (step 2) interacts with factors at the Cap.
        5.    eIf4 component and factors from the 5' untranslated region melt any stem-loops in the
               5'untranslated region. Requires ATP.
        6.    Small subunit complex scans along until it finds the first AUG. This is called
                ribosome scanning.
        7.    Initiator tRNA anticodon binds to AUG codon.eIF6 leaves the small subunit.
        8.    Large ribosomal subunit joins creating the functional ribosome with a P, A and E site.
        9.    eIF2-GDP is released and binds to eIF2B. GDP is displaced and GTP binds. This
                can now interact with initiator tRNA and start another cycle of initiation.
        10.    eIF2 is a major target for controlling translation in the cell.
                a.    When phosphorylated, it permanently binds to eIF2B. This sequesters the eIF2B and does not allow
                        further eID2-GTP active forms to be made. Lowers translation overall.
        11.    Internal Ribosome Entry can occur on some eukaryotic mRNAs that are under
                 tight control of translation (for example growth factors, etc.).
                a.    IRES sequences just upstream of an AUG are recognized by the initiation
                      complex which then moves to the AUG and initiates there. A truncated eIF4G binds to a stem-loop made
                        made by the IRES sequence and facilitates the attachment of the initiation complex.
   B.    Elongation
        1.    eEF1alpha bound to GTP binds to tRNA.
        2.    This complex moves to the A site of the ribosome.
        3.    If the anticodon on the tRNA is correct for the codon, it is held there long enough for
        4.    GTP to be hydrolyzed to GDP causing the eEF1alpha to disconnect from the tRNA and leave
        5.    The energy released in step 4 stably attaches the tRNA to the A site.
        6.    Peptidyl transferase activity (probably rRNA in the ribosome) catalyzes the breaking of
                the bond holding methionine to the initiator tRNA and using the energy released to
                attach the methionine to the amino acid attached to the tRNA at the A site by a
                peptide bond.
        7.    With the help of eEF2 bound to GTP, the ribosome moves one codon along the mRNA so
                that the tRNA is now at the P site and the A site awaits another tRNA complex. The exiting tRNA from
                the P site transiently is at the E site. Then leaves.
                This is called translocation.
        8.    The process repeats itself over and over until a stop codon is at the A site.
        9.    The eEF1alpha bound to GDP (see step 1) is regenerated to the GTP bound form by
                eEF1b binding to the GDP bound form, causing its displacement and allowing the GTP
                to bind. It can now interact with another tRNA.
        10. This mechanism provides a kinetic proofreading that insures that only the correct tRNA
               will remain at the A site long enough to allow the above events to happen before a
               peptide bond is made.

    C.    Termination
        1. When a stop codon is at the A site of the ribosome,  RF1 (or 2, depending on the stop codon) binds
            there, interacting directly with the stop codon.RF3-GTP binds the A site elsewhere.
        2.    Peptidyl transferase breaks the attachment between the polypeptide and the tRNA at the
               P site. Water enters.
        3.    The polypeptide is released with an intact COOH terminus; the tRNA at the P site exits
               and RF3 hydrolyzing GTP to GDP. The entire ribosomal
                complex comes apart.

    D.    Additional events of note
        1.    Efficiency of translation is increased because the poly A tail of the mRNA binds
           poly-Abinding protein 1 (PAB1). This interacts directly with the eukaryotic initiation factor
           at the Cap, facilitating the entry of the small ribosome initiation complex onto the mRNA to
            start a new round of initiation.

    E.    Prokaryotic Differences (Not covered this year. Not on test)
        1.    Initiation and setting of the reading frame can occur in more than one place (polycistronic)
            a.    uses Shine-Delgarno interactions between mRNA codons before the AUG interacting
                    with ribosomal RNA sequences.

    F.    Control at the level of translation
            a.    The ferritin mRNA translation is controlled at the level of initiation.
                -The stem-loop in the 5'UTR contains an Iron Response Element that can be bound by
                the IRE binding protein aconitase if concentrations of iron are low in the cell.
                -This prevents the small subunit inititation complex from scanning along to find AUG after
                the Cap.
                -Translation cannot be initiated.
                -If, however, iron concentrations are high, the ferritin should be made to store the excess
                  iron and the IRE binding protein will not be able to bind and the stem-loop can be
                  melted. Translation can be initiated.

     G.    Selenocysteine is found in a few proteins. (Not covered on test this year).
            a.    tRNA for selenocysteine has serine added first. Chemically altered to selenocysteine.
            b.    Interacts with a special translation factor-GTP which binds to the A site upstream from a signalling
                    stem-loop structure in the mRNA.
            c.    The A site has a UGA (usually a stop codon) at it in the mRNA, but now is used as the codon for
                 the selenocysteine tRNA anticodon. Translation continues.

V.      Control at the level of mRNA stability

        A.    The 3' untranslated region of eukaryotic mRNAs can contain repeated AU sequences
               that signal the poly-A tail to be rapidly degraded by nucleases.
            1.    This causes these mRNAs to have very short half-lives
            2.    The proteins coded for by these mRNAs are usually central to growth regulation for
                    the cell.
            3.    If a long-lived mRNA like b-globin has its 3'UTR replaced by these sequences
                    from a short-lived mRNA, it becomes short-lived also.

        B.    Example we looked at was the mRNA for the transferin receptor.
            1.    Has several AU repeats in its 3'UTR.
            2.    Allows the formation of stem-loops that contain an iron-response element (IRE).
            3.    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 sigalling for degredation.
                   -Therefore, translation will occur and more transferin receptor will be made.
                   -This will allow more iron to be brought into the cell.
                    -When iron levels are high, no more iron entry is needed. Aconitase does not bind and the mRNA
                        degrades.