16.0 Introduction

1. Genes Are Not All Expressed at Once
1. Different Genes Expressed at Different Times
2. Genetic "Score" Is that of Regulatory Genes fig 16.1

1. An Overview of Transcriptional Control
1. Regulating Promotor Access
1. One way to control regulation of transcription
1. RNA polymerase must have access to DNA helix
2. Must be able to bind to gene's promotor

2. Can also regulate by affect ability of RNA polymerase to bind to promotor
1. Promotor binding sites 10 to 15 nucleotides long
2. Hundreds characterized, each is specific
3. Binding to promotor has one of two results
1. Blocks transcription by interfering with RNA polymerase
2. Stimulates transcription by facilitating polymerase binding

2. Transcriptional Control in Prokaryotes
1. Bacteria must exploit transient resources of environment
2. Gene control adjusts cell's activities to fit environment
3. Changes in gene expression are generally reversible

3. Transcriptional Control in Eukaryotes
1. Cells need to be protected from transient environmental changes
2. Prefer constant conditions
3. Homeostasis
1. Maintenance of constant internal environment
2. Hall mark of multicellular organisms

4. Individual cells respond to signals in immediate environment
1. Alter their gene expression
2. Also participate in regulating body as a whole

5. Changes in gene expression produce variety of results
1. Compensate for changes in body's physiological condition
2. Ensure that correct genes are expressed in development

6. Genes must be transcribed in careful order, for specific time
1. Many genes activated only once
1. Example: Stem cells
2. Develop onto differentiated tissues via strict genetic program

7. Changes in gene expression serve needs of whole, not individual cell

4. Posttranscriptional Control
1. Gene expression regulated at many levels
2. Most common is transcriptional control
3. Less common is posttranscriptional control
1. Control occurs after transcription has occurred
2. Influences mRNA produced from genes
3. Influences activity of proteins encoded by mRNA

1. How to Read a Helix Without Unwinding It
1. Looking into the Major Groove
1. DNA helix does not need to unwind for recognition by proteins
2. Proteins bind to outer surface, at edges of base-pairs
3. Major groove
1. Deeper of two helical grooves that wind around DNA molecule fig 16.2
2. Nucleotide chemical groups are accessible, make unique patterns

2. DNA Binding Motifs
1. Protein-DNA recognition is being actively studied
2. Proteins are unique, less variability where it actually binds to DNA
3. Several different groups called structural or DNA-binding motifs

2. Four Important Motifs
1. The Helix-Turn-Helix Motif
1. Most common DNA-binding motif
2. Two alpha-helical segments linked by short nonhelical segment fig 16.3
1. Segments are at right angles to one another
2. Recognition helix fits into major groove of DNA
3. Other helix butts up against outside of DNA
4. Ensures proper positioning of recognition helix

3. Regulatory sequences recognized by this motif occur in symmetrical pairs
1. Sequences separated by distance equal to one turn of helix, 3.4 nm fig 16.4
2. Two sites doubles contact zone between protein and DNA

2. The Homeodomain Motif
1. Special class of helix-turn-helix motif plays important role in development
2. Present in wide variety of eukaryotes, including humans
3. Discovered in homeotic mutants of Drosophila
1. Mutations alter how body parts are assembled
2. Mutant genes encode regulatory proteins that initiate key stages in development by binding to developmental switch-point genes
3. Proteins have nearly identical sequence of 60 amino acids
4. Sequence called homeodomain fig 16.5b
5. Center of homeodomain is helix-turn-helix motif
6. Ensures motif is always presented to DNA in same way

3. The Zinc Finger Motif
1. Uses atoms of zinc to coordinate binding to DNA fig 16.5c
2. In one form zinc links alpha-helical segment to beta-sheet segment
1. Helical segment fits into major groove
2. Often occurs in clusters
3. Beta-sheet spaces helical segments to each contacts major groove
4. Binding stronger with more zinc fingers

3. Other forms replace beta-sheet with another helical segment

4. The Leucine Zipper Motif
1. Two protein subunits create a single DNA-binding site
1. Subunits interact at leucines forming Y-shaped molecule
2. Arms of Y fit into major groove fig 16.5d

2. Allow great flexibility in controlling gene expression

1. Controlling Transcription Initiation
1. Repressors Are Off Switches
1. Only genes that are directly needed are transcribed
1. Others held in reserve
2. Make enzymes to degrade a type of food only when it is present

2. Example: Tryptophan-producing (trp) genes in E. coli
3. Operons
1. Cluster of five genes manufacturers tryptophan
2. Unit called an operon, produces long strand of mRNA
3. RNA polymerase binds to promoter at first gene, transcription ensues
4. Regulators shut off transcription by binding to operator site in front of promotor

4. Tryptophan present, trp genes shut off by repressor
1. Helix-turn-helix regulatory protein, binds to trp promoter fig 16.6
2. Repressor at promoter prevents binding of RNA polymerase
3. Repressor can't bind unless first bound to two tryptophans
4. Tryptophan alters orientation of helix-turn-helix in repressor
5. Recognition helices fit into DNA major groove fig 16.7,8

5. Synthesis of tryptophan tied to absence of it in environment
1. Without it nothing activates repressor, transcription ensues
2. With it, it binds to repressor, blocks transcription, halts synthesis of tryptophan

2. Activators Are On Switches
1. Some gene promotors constructed to be poor RNA polymerase binding sites
2. Transcription of these genes rarely occurs unless promotor can bind better
1. Requires transcriptional activator
2. Binds to DNA nearby
3. Holds polymerase against promoter, RNA polymerase binds better

3. Example: Catabolite activator protein (CAP) of E. coli fig 16.9
1. Initiates transcription of genes to utilize food when glucose absent
2. Decreasing glucose leads to increase in intracellular cyclic AMP (cAMP)
3. cAMP binds to CAP, protein changes shape
4. CAP's helix-turn-helix motif binds to DNA near several promoters
5. Promoters activates, genes transcribed

3. Combination of Switches
1. Sophisticated systems created by combining On and Off switches
2. Example: lac operon of E. coli fig 16.10
1. Produces three proteins that import disaccharide lactose
2. Breaks it into two monosaccharides, glucose and galactose

3. The activator switch
1. lac operon has two regulatory sites
2. CAP site adjacent to lac promoter fig 16.11
1. Ensures genes not transcribed when glucose is present
2. If glucose absent, high levels of cAMP in cell
3. cAMP binds to CAP,CAP binds to DNA, promoter functional
4. If glucose present, levels of cAMP are low
5. CAP prevented from binding to DNA, lac promoter not functional

4. The repressor switch
1. Operator is second regulatory site, adjacent to promoter fig 16.12
2. lac repressor binds to operator, only when lactose absent
1. Repressor covers part of promoter when bound to operator
2. RNA polymerase can't bind, lac genes not transcribed

3. Cell doesn't transcribe genes to make product it doesn't need
4. If lactose present, lactose isomer binds to repressor
1. Repressor binding motif twisted away from major groove fig 16.13
2. Repressor can't bind to operator, RNA polymerase can bind to promoter,
   transcription of lac genes ensues
3. Transcription of operon induced by presence of lactose

5. Lactose utilizing proteins made when lactose present, glucose not present
1. Metabolic decision to produce only what cell needs
2. Conserves resources fig 16.14

1. Designing a Complex Gene Control System
1. Eukaryotic Systems Inherently More Complex than Prokaryotic Systems
1. Prokaryotic control limited to number of switches that fit around promotor
2. In eukaryotes many genes interact with each other
1. Requires more interacting elements than can fit around one promotor tbl 16.1
2. Limitation overcome by exerting control through distant sites

3. Control from a distance mechanism includes two non-bacterial features
1. Set of proteins that help bind RNA polymerase to promotor
2. Modular regulatory proteins that bind to distant sites

4. Features produce flexible control system

2. Eukaryotic Transcription Factors
1. Assists binding of RNA polymerase to promotor tbl 16.1
1. Assembles on promotor
2. Guides and stabilizes binding of polymerase fig 16.15
3. Assembly begins 25 nucleotides upstream from start site
4. Binds to short TATA sequence
5. Other factors bind making transcription factor complex to capture RNA polymerase
6. May then phosphorylate bound polymerase
1. Disengages it from complex
2. Frees polymerase to begin transcription

2. Several transcription factors provides numerous points for control fig 16.16
3. Transcription inhibited by
1. Anything that reduces availability of any factor
2. Anything that limits its ease of assembly into complex

3. Enhancers
1. Composed of two distinct modules (domains)
2. DNA-binding domain: Attaches protein to DNA at specific site
3. Regulatory domain: Interacts with regulatory proteins
4. Modular design uncouples regulation from DNA binding
1. Allows binding at one site and regulation at more distant site
2. Enhancers are distant regulatory sites
3. Occur occasionally in bacteria, regularly in eukaryotes fig 16.17

5. Mechanism of distant action fig 16.18
1. DNA loops around to position enhancer near promotor
2. Regulatory domain brought into direct contact with transcription complex attached to promotor

6. Permits large number of different sequences scattered around DNA to influence one gene

2. The Effect of Chromosome Structure on Gene Regulation
1. Review of Eukaryotic DNA Packaging
1. Nucleosomes formed by wrapping DNA around histone proteins fig 16.19
2. Strand of nucleosomes twisted into 30 nm filaments

2. Promotor Blocking by Nucleosomes
1. Histones over promotors block assembly of transcription factor complexes
1. Transcription factors unable to bind to nucleosome-packaged promotor
2. Nucleosomes may prevent continuous transcription initiation

2. Activators and RNA polymerase not inhibited by nucleosomes
1. Regulatory domains of activators plus enhancers displace histones
2. Displacement of histones required for assembly of complex
3. With transcription, RNA polymerase pushes histones aside

3. DNA Methylation
1. Methylation once thought to regulate gene transcription in vertebrates
2. Cytosine and uracil can be methylated, doesn't affect guanine or adenine fig 16.20
3. Many inactive mammalian genes are methylated
1. Once though to be cause of inactivation
2. Now thought to simply block accidental transcription of "turned-off" genes
3. Ensures that once a gene is turned off, it stays off

3. Post-Transcriptional Control in Eukaryotes
1. Gene Transcription Can Be Regulated at Points After Transcription fig 16.21
1. All serve as control points for some eukaryotic genes
2. mRNA sequences recognized by regulatory proteins, RNA molecules

2. Processing of the Primary Transcript
1. Exon-intron patchwork structure of eukaryotic genes
1. Numerous exons are short, expressed, coding sequences
2. Introns are lengthy, intervening noncoding sequences

2. Introns removed by enzymes during RNA processing or RNA splicing
3. Process of RNA splicing
1. Require small ribonucleoproteins (snRNPs) particles
2. Particles reside inside cell, composed of small nuclear RNA (snRNA or snurps)
3. Introns ends marked by sequences of nucleotides complementary to snRNA sequences
4. Multiple snRNPs combine to form larger complex called spliceosome
5. Intron is loops out and excised fig 16.22, 23

4. RNA splicing provides point for control of gene expression
1. Exons can be spliced together in different ways
2. Allows variety of polypeptides to be made from single gene
3. Common in insects and vertebrates

5. Expression regulated by changing splicing event over development or in different tissues

3. Transport of the Processed Transcript Out of the Nucleus
1. Processed mRNA transcripts transported out through nuclear pores
2. Active process requires recognition by pore receptors
1. Poly-A tail at 3' end plays a role in this recognition
2. Transport doesn't occur if any splicing enzymes are still attached
3. Ensures partially processed transcripts are not transported

3. Little evidence of regulation at this point
1. 10% of transcribed genes are exons, 5% reaches cytoplasm
2. Only half of primary transcripts leave nucleus
3. Unknown whether this is under selective control

4. Selecting Which mRNAs Are Translated
1. Translation of processed mRNA transcripts
1. Involves complex of proteins called translation factors
2. Gene expression regulated by modification of these factors

2. Translation repressor proteins shut down translation
1. Bind to beginning of transcript, prevent attachment to ribosome
2. Example: Ferritin shut off by aconitase repressor protein
1. Aconitase binds to 30 nucleotide sequence of ferritin mRNA
2. Forms stable loop to which ribosomes cannot bind
3. Presence of iron causes aconitase to dissociate
4. Increases ferritin production one hundredfold

5. Selectively Degrading mRNA Transcripts
1. Most eukaryotic mRNA transcripts are very stable
2. Regulatory protein and growth factor transcripts are less stable
1. Instability due to specific sequences at 3' end
2. Sequences make them targets for mRNA degrading enzymes

3. Examples
1. Sequence of A and U nucleotides near 3' poly-A tail
1. Promotes removal of tail
2. Destabilizes mRNA

2. Endonuclease recognition sites sequences, cause transcripts to be digested quickly

4. Regulatory transcript instability facilitates rapid alteration of level