Signals in DNA tell RNA polymerase where to start and stop transcription

• Synthesis starts at a promoter, a conserved sequence 5’ of a gene.

• Chain elongation occurs until RNA polymerase encounters a termination sequence on the DNA.

• RNA polymerase releases the single stranded RNA and the double stranded DNA template upon encountering a termination sequence.

• This slide depicts a bacterial RNA polymerase. Transcription initiation in eukaryotic cells is more complicated.

Promoter and terminator sequences in bacteria

Propose a mechanism by which the highlighted areas could cause transcription to stop.

5) The first two sequences are inverted repeats of each other, thus a hairpin, which is a signal for termination.

Clicker question

What is the same in all* somatic cells of your body?

1) DNA2) RNA3) Proteins4) Lipids5) Carbohydrates6) Lysosomes

* Some immune cells are interesting exceptions.

The DNA in differentiated cells contains all the instructions to make a new organism

• Cell types differentiate during development of a multicellular organism to become specialized (e.g., muscle, nerve, brain, blood cells are different from each other), but they contain the same DNA.• What is different is that they express different genes, so they accumulate different sets of RNA and protein.

This is reproductive cloning -- how Dolly the Sheep was created. In theory, this procedure could be done in humans, but it would be unethical because most of the embryos are abnormal.

Cells regulate expression of proteins at many levels

We will discuss transcriptional control.

Transcription is controlled by proteins binding to regulatory sequences on

DNA

• Almost all prokaryotic and eukaryotic genes have regulatory DNA sequences that are used to switch the gene on or off.• Gene regulatory proteins bind to the regulatory DNA sequences.

- Repressors prevent transcription- Activators promote transcription

• Early studies of gene regulation involved bacteriophage and bacterial genes, which have simpler regulatory regions than eukaryotic genes.

Repressors block RNA polymerase -- prevent transcription initiation

Activators interact with RNA polymerase to help

initiate transcription

Bacteriophages (bacteria eaters) --Viruses that infect bacteria

• After infection, phage can live in one of two states

– Lytic: phage degrades host DNA, hijacks host cell machinery to produce many new phage. When cell is depleted, phage lyse their host bacterium to release new phage.– Lysogenic: phage DNA integrates into host chromosome. Prophage (phage DNA) duplicated every time host divides. Stress to host causes excision of prophage DNA and entry into lytic phase.

• Classic example of transcriptional regulation to turn on and off genes

Viruses that infect bacteria (bacteriophage or phage) look like syringes

Levine, A. “Viruses” p. 34

Bacteriophage infection

www.seyet.com/t4_academic.html

Growth of phage

Figure 1.2. Mark Ptashne. A Genetic Switch: Gene Control and Phage

Genetic switch between two states in phage

Lysogenicstate

(cI protein also called repressor)

Lysogenic stateLysogenic genes on; lytic genes off

repressor (cI protein) blocks transcription of Cro (a lytic gene) and activates transcription of its own gene.

Phage genes are off Repressor gene is on

”repressor” acts as both a repressor and an activator

A repressor physically blocks RNA pol from initiating transcription.An activator interacts with RNA pol to promote transcription.

Lytic stateLytic genes on; lysogenic genes off

Repressor gene is off Phage genes are on

Cro repressor binds to OR3, preventing transcription of repressor (cI protein) and other lysogenic genes. RNA pol can now transcribe Cro gene.

Lytic state: UV light or other inducer causes phage genes to be turned on and new phage to be produced, which results in lysis of the host cell. Viral repressors (green spheres) prevent transcription of lysogenic genes, but not lytic genes.

Repressor gene is off Phage genes are

on

A genetic switch in prokaryotes (lysis versus lysogeny of a virus) is controlled by regulation of transcription

Lysogenic state: Bacteriophage genome is integrated into bacterial genome. Bacteriophage is dormant and phage genes are not expressed. Viral repressor proteins (red dumbbells) block transcription of lytic genes and activate transcription of repressor genes.

Phage genes are off

Repressor gene is on

Transcriptional regulators (repressors, activators) are proteins that recognize specific DNA sequences

Beamer & Pabo, 1992, J. Mol. Biol. 227: 177.

• -helices of proteins fit into the major groove of DNA.

• Amino acid sidechains from the protein make specific contacts with exposed edges of basepairs.

Structure of bacteriophage lambda repressor

Sequence-specific recognition of DNA by proteins Seeman et al. (1976) PNAS 73:804-808

Majorgroove

Minorgroove

Major groove

Minor groove

A A D A D A

Clicker question

Many DNA binding proteins (including restriction enzymes) recognize palindromic* sequences of DNA. What does binding to a palindromic DNA sequence imply about the structure of a DNA-binding protein?

1) It is a dimer with translational symmetry2) It is a dimer with two-fold rotational symmetry3) It is a dimer with mirror (inversion) symmetry4) It is a trimer with translational symmetry5) It is a trimer with three-fold rotational

symmetry6) It is a trimer with three-fold mirror symmetry

*Palindrome examples: MADAM I’M ADAM orGAAGCTCGTACGAGCTTC CTTCGAGCATGCTCGAAG

Clicker question

What does binding to a palindromic DNA sequence imply about the structure of the DNA-binding protein?

1) It is a dimer with translational symmetry2) It is a dimer with two-fold rotational symmetry3) It is a dimer with two-fold mirror symmetry

*Palindrome examples: MADAM I’M ADAM orGAAGCTCGTACGAGCTTC CTTCGAGCATGCTCGAAG

Transcriptional regulation of eukaryotic genes is more complicated than regulation of prokaryotic

genes

• Basal promoter elements (e.g., TATA box: 25 bp upstream of transcription start site).

• Promoter proximal element. 100-200 bp long -- close to site of transcription initiation. Contains sequences recognized by different transcription factors.

• Enhancer elements. Can be a few thousand to 20,000 bp upstream or downstream from the initiator site.

Transcriptional activation involves interactions over long stretches of DNA

• TATA box binding protein (TBP) binds to RNA polymerase and other proteins to form pre-initiation complex.

TBP

TATA-binding protein (TBP)

Transcriptional activation involves interactions over long stretches of DNA

• TATA box binding protein (TBP) binds to RNA pol and other proteins to form pre-initiation complex.

• Pre-initiation complex interacts with different specific transcription factors bound to promoter proximal elements and enhancer elements.

• Each gene in every cell has same DNA control sequences, but not every cell has complete set of DNA binding proteins to turn on every gene.

NFB

• NFB proteins are cytoplasmic. Inactive when bound to I-B.

• Inducers (stress, infection, etc.) trigger dissociation and degradation of I-B, then NFB enters nucleus and activates genes.

• NFB binds to B sites in the enhancer regions of genes involved in cellular defense mechanisms and differentiation.

• NFB induces transcription of HIV viral RNA by the host cell RNA polymerase.

• David Baltimore’s lab at Caltech works on NFB.

Stress signal comes from outside the cell.

A signaling cascade in the

cytoplasm results in

degradation of I-B.

NFB enters the nucleus

and activates genes.

Crystal structure a piece of NFB bound to a B site on DNA

• Structure resembles butterfly with protein domains as wings attached to cylindrical body of DNA.

• Contacts with DNA formed by loops between -strands. No helical or sheet structure at recognition surface.

Ghosh et al., 1995, Nature 373: 303-310; Müller et al., ibid, 311-317

How can you tell when/where a gene is being expressed?

Hybridization can be used to locate when/where a gene is expressed or compare genomes

• Take advantage of ability of DNA to pair selectively with a second strand of complementary nucleotide sequence.

• Denature DNA (heat or pH) to separate strands, then slowly reverse to allow double helices to reform (hybridization or renaturation).

• Any two complementary single-stranded nucleic acid chains can hybridize (DNA/DNA, RNA/RNA, RNA/DNA). [HIV reverse transcriptase makes cDNA, or complementary DNA, from a single-stranded RNA template.]

Figure 10-12, Little Alberts

1. The hydrogen bonds that form double-stranded DNA are easily disrupted by heating.

2. Some dyes fluoresce when they bind to double-stranded DNA.

Physical Chemistry of DNA Hybridization

Studied at Caltech in 60s and 70s

by Norman Davidson

Can use hybridization to search for gene expression

• Make a ssDNA probe (can be synthesized) to detect complementary sequences of interest.

• Can probe for normal versus mutant forms of a gene (e.g., sickle-cell anemia gene; cancer-predisposing genes).

• Can probe cDNA from cells at a particular developmental stage or from a tissue to see when or where a gene is expressed.

• Can do hybridizations in situ (in place) to locate nucleic acid sequences in cells, organisms (below left) or on chromosomes (below right).

Whole mount zebrafish in situ hybrization kit

2 m

FISH -- FluorescenceIn Situ Hybridization

DNA microarrays to evaluate gene expression

• Array thousands of DNA oligonucleotides (probes) in known locations, each is a different sequence

• Add target cDNA (complementary DNA made from transcribed RNA) (target) to hybridize under high-stringency conditions

• Probe-target hybridization detected by and quantified by fluorescence

Commercial Gene Chips

Probes attached covalently to a chemical matrix on a solid surface (quartz). Made using photolithography.

Applications

• Gene expression profiling -- monitor mRNA for thousands of genes to study effects of:

– different stages in development or differentiation– disease (cancer)– infection

• Compare genomes in different organisms• Identify single nucleotide polymorphisms (SNPs) for genotyping, forensics, to study predisposition to disease, find mutations in cancers• Alternative splicing detection -- use probes for predicted exons of a gene

Dual Color Microarray experimen

t

n.m.wikipedia.org/wiki/DNA_microarray

Red Fluorescent Probes Green Fluorescent Probes

~40,000 probe spotted oligo microarray

n.m.wikipedia.org/wiki/DNA_microarray

Yellow -- merge of red and green fluorescence