BI 314 - TA contact information:

Brian Dixon                                                                dixonb@bcc.orst.edu
Weniger 538/540                                                        737-8003
Office hours: MTW 9-10 or by appointment

Jie Hao                                                                        haoj@bcc.orst.edu
Cordley 4068                                                                737-5295
Office hours: Th 2-5, or by appointment

CD-ROM Quiz Questions to Study for Exam II on Wednesday, 7 February, 2001:

Ch. 3 = 4-9
Ch. 4 = 2-4, 7,8   (note: Q#1 - the correct answer is now wrong, now know that correct answer = 30,000-35,000 genes)
Ch. 5 = 1-9
Ch. 6 = 1-5, 7-12

Review session = Monday, 5 February from 5-7 PM, Cordley 1109
 
 

Chapter 6 - RNA SYNTHESIS AND PROCESSING




Characteristics of cells determined both by genes it inherits and when or if those genes are expressed

        - when a particular protein is needed by the cell, appropriate stretch of the
                DNA molecule (gene) is copied into RNA

    Transcription = RNA copy made from DNA sequence

        - produces RNA complementary to 1 strand of DNA

        - primary level at which gene expression is controlled

        - many RNA copies can be made from a single gene

        - each RNA molecule can direct the synthesis of many identical proteins

        - allows amplification of the amount of protein made

                - lots of some, tiny amounts of others

        - cell can regulate expression of each gene according to needs of moment

    Translation = RNA message converted to protein sequence

          - RNA determines linear sequence of amino acids

--> determines properties and functions of proteins TRANSCRIPTION IN PROKARYOTES

        - more simple process, everything is in one compartment

                - but, fewer levels of control

        - studying prokaryotes (esp. E. coli) has laid foundation for studying
                    more complex regulation in eukaryotic cells

RNA polymerase

        - catalyzes polymerization of ribonucleoside 5ítriphosphates (NTPs)
                    from a DNA template

        - does not need primer to start

        - initiates de novo @ specific sites @ beginning of genes

        - does not have proof-reading function (1 mistake/104 nucleotides)

                - doesnít have to be as accurate, no permanent storage function

        - moves stepwise along DNA, unwinding helix just ahead

                - growing RNA chain is extended 1 nucleotide at a time

                - adds nucleotides 5í-3í using nucleoside triphosphates for energy

        - almost immediate release of RNA strand from DNA as it is synthesized

                - allows multiple RNAs to be synthesized at once

        - fast - medium-sized gene (~1500 bp) requires ca. 50 sec. for a molecule of
                        RNA polymerase to transcribe it

                - w/ 15 polymerases working at once, can make >1000 transcripts/min
 

Signals in DNA tell RNA polymerase where to start and finish

        promoter = DNA sequence indicating start point for RNA synthesis

            - made up of two 6 base pair sequences at -10 and -35 upstream of
                      transcription start site = beginning of gene where RNA nucleotides
                        start to be synthesized

            - RNA polymerase randomly collides w/ DNA, sticks weakly, then slides
                        along DNA rapidly until it hits promoter

            - polymerase can recognize a promoter even when double helix is intact

                    - contacts exposed portions of bases in grooves

            - promoters are asymmetrical and bind polymerase only in one orientation

                    \ can only transcribe one strand

            - transcription can proceed only 5í-3í

            - direction of transcription with respect to chromosome varies from
                       gene to gene

            - canít get transcription w/o a promoter

polymerase binds tightly to promoter DNA
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opens double helix immediately in front of it
(unwinds and rewinds as it goes)
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one exposed strand acts as template
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first 2 nucleotides joined
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chain elongation until polymerase encounters terminator (stop site)
-one type =stem-loop structure formed by
complementary base pairing
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polymerase stops, releases DNA and RNA chain








        sigma (s) factor = subunit of bacterial polymerase which recognizes promoter

                - binds specifically to -35 and -10 sequences

        - released once 10 nucleotides of RNA synthesized

        - reassociates w/ polymerase once it is released at terminator

        - searches for new promoter
 
 

Similarities between transcription and DNA replication

        - begins w/opening & unwinding small portion of DNA helix

        - 1 of the 2 strands of DNA acts as template for RNA synthesis

        - nucleotide sequence of RNA chain determined by complementary
                    base pairing of incoming ribonucleotide

        - incoming ribonucleotide covalently linked to growing chain
                    catalyzed by RNA polymerase

  Differences between transcription and DNA replication:

        - RNA strand does not remain H-bonded to DNA template

        - DNA helix reforms and displaces the RNA strand

        - RNA molecule is single-stranded and shorter

                - DNA (human chromosome) = 250,000,000 nucleotides long

                - RNA = 3,000 or less
 
 

Control of Transcription

        Mechanism necessary for transcribing genes only when needed

             - e.g. enzymes necessary for utilizing lactose synthesized only when
                            lactose present

            - ß-galactosidase, lactose permease, and transacetylase transcribed
                        together as operon

       operon = group of adjacent genes transcribed as a single mRNA

       operator = regulatory sequence that controls transcription of an operon

        repressor = protein that blocks transcription when bound to an operator

            - in the case of the lac operon, lactose binds to the repressor,
                    preventing it from binding to the operator

                    - lac operator = 30 bp just before transcription start site

                    - binding of repressor keeps RNA polymerase from binding
 

       central principle of gene regulation = control of transcription is
                mediated by interactions of regulatory proteins with specific
                DNA sequences

              - true for both prokaryotes and eukaryotes

        cis-acting control elements = DNA regulatory sequences,
                    e.g. operator

                    - affect expression only of linked genes on the same DNA

        trans-acting factors = transcription regulatory proteins,
                    e.g. lac repressor

                    - can affect expression of genes located on multiple chromosomes

                    - can be repressors or activators of transcription

  CONTROL OF TRANSCRIPTION IN EUKARYOTES

        Regulation of transcription much more complex in eukaryotic cells:

                - eukaryotic cells contain multiple types of RNA polymerase that
                    transcribe distinct classes of genes

                - eukaryotic RNA polymerases need to interact w/variety of additional
                    proteins to initiate transcription

Eukaryotic RNA polymerases

3 distinct nuclear RNA polymerases:

        RNA polymerase I - transcribes 3 largest rRNAs (28S, 18S, 5.8S+ a few small RNAs)

        RNA polymerase II - transcribes protein-coding genes = mRNAs

        RNA polymerase III - transcribes tRNAs and the remaining small RNAs

                - share 5 common subunits (each contains 8-14 subunits, total) and
                    all require additional proteins to initiate transcription

        general transcription factors = additional proteins required to
                initiate transcription by eukaryotic RNA polymerase II

                - constitute part of basic transcription machinery

                - in vitro, need 5 general transcription factors for RNA polymerase II
                        binding to TATA box (TATAA) 25-30 nucleotides upstream of
                        transcription start site:

                    TFIID = TATA-binding protein (TBP) + associated factors (TAFs)

                        - recognizes and binds TATA box ? bends DNA 110º

                    TFIIB - binds to TBP

                    Polymerase and TFIIF then bind, followed by TFIIE and TFIIH

                        - Then transcription is initiated

        TBP = TATA-binding protein = also required for initiation of
                        transcription by RNA polymerase I and III

        RNA polymerase I transcribes genes for large 45S pre-rRNA,
                    which is then processed to 28S, 18S, and 5.8S rRNAs

                - UBF (upstream binding factor) and SL1 (selectivity factor 1, contains TBP)
                            bind to 150 bp promoter, recruit polymerase I

        RNA polymerase III binds to promoters that lay within transcribed sequences

                - e.g. 5S rRNA gene = TFIIIA binds downstream of transcription start site,
                        followed by TFIIIC, TFIIIB (contains TBP), and polymerase III

                - tRNA genes = TFIIIC binds to promoter -->TFIIIB --> polymerase

Promoter and Enhancers = cis-acting sequences that regulate expression
        of adjacent eukaryotic genes

        - usually located upstream of TATA box

       core promoters usually located close by

       enhancers can be located up to several kilobases away from
                transcription start site

            - location not important - can stimulate transcription from upstream,
                    downstream, in forward or backward orientation

            -stimulate other genes when put in front of their promoters

            - bind transcription factors, then regulate RNA polymerase

            - DNA looping brings transcription factor into proximity of RNA
                    polymerase/general transcription factor complex

            -transcription factors confer developmental and environmental
                    signals

            - enhancers usually contain multiple functional sequence elements

                    - bind different transcriptional regulatory proteins

                    - work together to regulate gene expression

        transcriptional activators = sequence-specific DNA-binding proteins
                that bind to regulatory DNA sequences and stimulate transcription

            - have two domains:

                - DNA-binding domain - recognizes specific DNA sequence

                        e.g. zinc finger domains

                                helix-turn-helix domains

                                leucine zipper domains

                                helix-loop-helix domains

                - protein:protein interaction domain - interacts with
                        transcriptional machinery

                        - thought to interact with TFIIB or TFIID and facilitate
                            assembly of transcription complex on the promoter

                        - different activators can bind different general transcription
                            factors, yielding synergistic stimulation of transcription

Eukaryotic repressors = also involved in regulating gene expression

        - some interfere with binding of activators or general transcription
                factors to DNA

                - have DNA-binding domain, but not activating domain

        - others contain discrete repression domains that inhibit
                transcription by interacting directly with activators or general
                transcription factors

    - important role for repressors = inhibition of expression of
            tissue-specific genes in the wrong cell types
 

Chromatin Structure Plays a Role in Regulating Transcription

    - Nucleosomes normally impede transcription in eukaryotic cells

            -  tightly-bound histones and nucleosome structure gets in the way of
                    transcription factors and RNA polymerase

            -  actively-transcribed genes found in decondensed chromatin

                    -  e.g. 10 nm fibers

    -  both activators and repressors not only interact with transcription
                    factors, but also induce changes in chromatin structure

    - acetylation (= addition of acetyl group) of histone tails by
                   coactivators (= histone acetyltransferase) plays
                    key role in gene expression

                            -  makes DNA more accessible

           -  histone acetylation associated w/transcriptionally active chromatin

                -  may weaken binding of histones to DNA or alter histone
                    interaction with other proteins

                -  allows binding of 2 non-histone proteins = HMG-14 and HMG-17
                            and other nucleosome remodeling factors

                        - alters nucleosome structure and facilitates binding of transcription factors

           -  not clear how activators and nucleosome remodeling factors are targeted to certain genes

    - deacetylation of histones by corepressors (=  histone deacetylases associated
                w/repressors) prevent accessibility of genes
 

Methylation of DNA = another general mechanism for control of transcription

        Cytosine residues can be modified by addition of methyl groups

                - occurs in C's that proceed G's in DNA chain

                -  correlated with reduced transcription of genes

        MeCP2 = protein that binds to methylated DNA, represses transcription

                -  functions in complex w/histone deacetylase

  RNA PROCESSING AND TURNOVER

        - in bacteria, as mRNA is transcribed, ribosomes immediately
                attach to free 5í end, translation starts

        - eukaryotic RNAs must be modified before they are functional

Eucaryotic RNAs undergo processing in the nucleus

        - bacteria and eukaryotes differ in how their RNA transcripts are
                processed before they can be used by the cell
    WHY?

        ribosomes = structure on which protein synthesis takes place

                - bacterial DNA is directly exposed to cytoplasm

                - in eukaryotes, DNA enclosed in nucleus - ribosomes are in cytoplasm
                        mRNA must be transported out through nuclear pores

                - before that, mRNA goes through processing steps

        primary transcript = pre-mRNAs = unprocessed RNA

            - in eukaryotic cells, pre-mRNAs are extensively modified before
                    export from the nucleus

        Processing steps:

              1.  RNA capping = modification of end of primary transcript

                        - addition of a 7-methylguanosine cap = GTP in reverse orientation
                                + methyl groups

                            -  occurs early in transcription

              2.  Polyadenylation - end trimmed by endonuclease, then
                       poly-A polymerase adds series of adenines (few hundred) = poly(A) tail

                       - signal = AAUAAA in mammals, cut occurs 10-30 bp downstream

                        -  both processes thought to increase mRNA stability, aid in export,
                            allows protein synthesis machinery to tell both ends are present
                            = message complete
 

                 3.  Introns removed by RNA splicing

transcription of entire length of gene = primary transcript
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capping and polyadenylation
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RNA splicing = intron sequences removed, exons joined together
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short RNA molecule w/uninterrupted coding sequence
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export out of nucleus
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translation into protein



How does cell know where to splice?

        - each intron has a few, short nucleotide sequences for cues

                - at or near each end of intron

                - very similar sequence in all introns

        - Introns removed by splicing enzyme complex = spliceosomes

              -  made of protein and RNA = snRNPs (small nuclear ribonucleoprotein particles)

                        - groups assemble on RNA, excise introns, religate RNA chain

        - excised intron released as lariat --> linearized and degraded

        - RNA in snRNPs recognizes nucleotide sequences that mark beginning
                    and branch point of intron and pairs them together using
                  complementary base pairing

                - brings the 2 ends of the intron together so splicing can happen

                - other proteins are also needed

        - existence of introns and exons may have facilitated evolution of
                new and useful proteins

                - numerous introns make genetic recombination between exons
                        of different genes more likely

                - can combine different parts to create new functionalities

                - many present-day proteins are made up of common sets of
                        protein pieces = protein domains

        - RNA splicing also allows different proteins to be made from a single gene

                - primary transcript is spliced in various ways to make diff. mRNAs

                - adds to enormous coding potential of the genome

                - but there is also a cost - cell has to maintain larger genome

        Some mitochondrial, chloroplast, and bacterial RNAs undergo self-splicing

                - the splicing reaction is catalyzed by the intron sequences = ribozymes

        Alternative splicing joins exons in various combinations

                - important for tissue-specific control of gene expression
 

 RNA Editing = processing events (other than splicing) that alter the
                            protein-coding sequences of some mRNAs

                    - e.g. addition of deletion of U's in some genes, or modification of
                                specific bases in some proteins

            -  can be used to give a different tissue-specific function to a protein
 

RNA Degradation

        mRNAs are eventually degraded by the cell

                - same mRNA molecule can be translated many times

                - lifetime of mRNA molecule affects amount of protein produced

                        - eventually degraded into nucleotides

                        - lifetime depends on nucleotide sequence (esp.
                                3í untranslated region), type of cell

                - most bacterial mRNAs are short-lived - ca. 3 min.

                - eucaryotic mRNAs can persist longer -->10 hr.

                - in general, long-lived mRNAs --> highly-expressed proteins
                      short-lived mRNAs --> low levels or must change rapidly in
                            response to extracellular signals

                - final amount of protein in cell depends on: efficiency of
                        transcription/translation, rate of protein/RNA degradation