1. Bio99A: Molecular Biology, Spring 2010 Part 2: Gene expression / transcription Hans-Ulrich Bernard (Uli Bernard)

2. Administrative issues: Instructor: Dr. Uli Bernard, Dept. Molecular Biology and Biochemistry + Progr. Publ. Health Office: 114 Sprague Hall Preferred Contact: discussion after lecture Other possibilities: visit my office Friday 11-12 a.m. (1/2 mile SW from here) Email: hbernard@uci.edu (but I cannot answer 400 emails per week)

3. I studied in Göttingen, Germany, apologies for my accent !

4. I worked for 15 years at the National University of Singapore, where I gave similar lectures as this one, but this is my first Bio99 lecture at UCI. This means: I know the science, but I am still learning about the student population.

5. Education systems vary: I passed elementary and high school, college and graduate school without taking a single multiple choice test. As a father of high school kids, I hate tests and I hate cramming. However, if you are in this room, you are here to become a biologist, and you really have to know this stuff. I will use about 80% material from “Tropp”, but part of the content is from elsewhere or by my own design. The test will be based on my slides, but read “Tropp” if you want to know more.

6. Overall outline of Bio99: Section 1: DNA and RNA, structure and enzymology Section 2: Organization of genes and genomes Expression of genes (transcription) Section 3: Expression of genes (translation) our topic

7. Individual topics: Lecture 1: Genes, genomes, gene expression. What is transcription initiation? Lecture 2: Promoters and RNA polymerases in prokaryotes Lecture 3: Lac operon, negative regulation Lecture 4: Lac operon, positive regulation, trp operon Lecture 5: Promoters and RNA polymerases in eukaryotes Lecture 6: Eukaryotic transcription factors and their binding sites Lecture 7: Regulated factors, response elements Lecture 8: Transcription in specific organs, differentiation, cancer Lecture 9: Histones and chromatin, epigenetic regulation of transcription

8. Lecture 1: Genes, genomes, gene expression, What is transcription initiation? Lecture 1: Genes, genomes, gene expression etc.

9. What is “gene expression”? Central concept “dogma” of molecular biology: Lecture 1: Genes, genomes, gene expression etc. Transcription + translation leads from storage form of genetic information (DNA) to functional proteins.

10. What is “gene expression”? Lecture 1: Genes, genomes, gene expression etc. Transcription + translation leads from storage form of genetic information (DNA) to functional proteins. This lecture series is about transcription.

11. Terminology: What is a gene? (simple model, appropriate for prokaryotes) Lecture 1: Genes, genomes, gene expression etc. Coding sequences plus flanking elements involved in expression.

12. Terminology: What is a gene ? (complex model more appropriate for eukaryotes) Introns (intervening sequences) Exons (coding sequences) Promoter region 3’ flanking region = 3’ non-coding region = downstream region determines transcription termination can contain regulatory elements 5’ flanking region = 5’ non-coding region = upstream region contains regulatory elements mRNA Transcription + splicing These segments have to be identified by sequence analyses and in functional studies, to detect meaning in a seemingly featureless DNA double helix. Lecture 1: Genes, genomes, gene expression etc.

13. What is an open reading frame (ORF) ? What is a cistron? What is a gene ? A segment of DNA that can encode a polypeptide sequence, i.e. is not interrupted by a termination codon. The term is used in pro- and eukaryotes and does not require the presence of an ATG. An ORF in prokaryotes which DOES contain an ATG. An mRNA can have one cistron (monocistronic) or several (polycistronic). Broader meaning than ORF and cistron. Definition can include flanking regulatory sequences. Also applies to non-coding sequences like rRNA and tRNA What is an operon? A set of genes, 2 or more, that are regulated and expressed together on a single mRNA (polycistronic mRNA). The genes in an operon have related functions. Lecture 1: Genes, genomes, gene expression etc.

14. Some number games: Size of proteins: Average 300 amino acid residues (but wide range of less than 100 to more than 1000) Average size of genes: 3 x 300 or 900 bp. Bacterium like E. coli has about 4300 genes = 4 million bp = actual genome size. All DNA is needed for coding of proteins. Humans have about 30,000 genes = 30 million bp, but the haploid human genome is 3 billion bp. Lots of junk in the human genome (remember, “junk” is not “garbage”). Lecture 1: Genes, genomes, gene expression etc.

15. What is a genome ? Genome size in nucleotide pairs for various organisms: about 4000 genes about 30,000 genes Lecture 1: Genes, genomes, gene expression etc. Sum of all genes plus non-coding sequences !

16. Gene + Genome properties of pro- vs. eukaryotes Prokaryotes: - most genes are contiguous, no introns - many transcription units have multiple genes (= cistrons organized in operons) - short non-coding regions - loose association of DNA and proteins - genome located in cytoplasm, forms nucleoid Eukaryotes: - many genes are not contiguous, have multiple exons - most transcription units have single gene - most DNA is non-coding - tight and complex association of DNA and proteins (= histones, form chromatin) - genome located in nucleus, transcription occurs in nucleus, translation in cytoplasm Lecture 1: Genes, genomes, gene expression etc.

17. Gene + Genome properties of pro- vs. eukaryotes Prokaryotes: - most genes are contiguous, no introns - many transcription units have multiple genes (= cistrons organized in operons) - short non-coding regions - loose association of DNA and proteins - genome located in cytoplasm, forms nucleoid Eukaryotes: - many genes not contiguous, have multiple exons - most transcription units have single gene - most DNA is non-coding - tight and complex association of DNA and proteins (= histones, form chromatin) - genome located in nucleus, transcription occurs in nucleus, translation in cytoplasm Lecture 1: Genes, genomes, gene expression etc.

18. Irrespective of pro- or eukaryote, genes can be on either strand of a double-stranded DNA Transcript map of protein coding transcripts in Adenovirus Lecture 1: Genes, genomes, gene expression etc.

19. Gene + Genome properties of pro- vs. eukaryotes Prokaryotes: - most genes are contiguous, no introns - many transcription units have multiple genes (= cistrons organized in operons) - short non-coding regions - loose association of DNA and proteins - genome located in cytoplasm, forms nucleoid Eukaryotes: - many genes not contiguous, have multiple exons - most transcription units have single gene - most DNA is non-coding - tight and complex association of DNA and proteins (= histones, form chromatin) - genome located in nucleus, transcription occurs in nucleus, translation in cytoplasm Lecture 1: Genes, genomes, gene expression etc.

20. Prokaryotic genomes are very compact: - very little space between genes - very little unfunctional (“junk”) DNA - existence of “operons” Lecture 1: Genes, genomes, gene expression etc.

21. Eukaryotic genomes are not compact Lecture 1: Genes, genomes, gene expression etc.

22. Gene + Genome properties of pro- vs. eukaryotes Prokaryotes: - most genes are contiguous, no introns - many transcription units have multiple genes (= cistrons organized in operons) - short non-coding regions - loose association of DNA and proteins - genome located in cytoplasm, forms nucleoid Eukaryotes: - many genes not contiguous, have multiple exons - most transcription units have single gene - most DNA is non-coding - tight and complex association of DNA and proteins (= histones, chromatin) - genome located in nucleus, transcription occurs in nucleus, translation in cytoplasm Lecture 1: Genes, genomes, gene expression etc.

23. Gene + Genome properties of pro- vs. eukaryotes Prokaryotes: - most genes are contiguous, no introns - many transcription units have multiple genes (= cistrons organized in operons) - short non-coding regions - loose association of DNA and proteins - genome located in cytoplasm, forms nucleoid Eukaryotes: - many genes not contiguous, have multiple exons - most transcription units have single gene - most DNA is non-coding - tight and complex association of DNA and proteins (= histones, form chromatin) - genome located in nucleus, transcription occurs in nucleus, translation in cytoplasm Lecture 1: Genes, genomes, gene expression etc.

24. Localization of genome and gene expression in prokaryotes versus eukaryotes Lecture 1: Genes, genomes, gene expression etc.

25. Why study regulation of gene expression? Lecture 1: Genes, genomes, gene expression etc. Bacterium: 4000 + genes Higher organism: 30,000 genes At any given time only a subset of these genes is expressed. And this subset determines: physiological properties morphological properties differentiation, health and disease, etc.

26. Gene expression: Be aware of the dimensions of the undertaking: 10 14 cells, each with the same 30.000 genes regulated gene expression Perfect human being Lecture 1: Genes, genomes, gene expression etc.

27. Imagine the challenge of gene expression : Even a small bacterial genome has thousands of genes, and is not just a circle but a really long string. The length of the DNA in each human cell is about 2 meters, 100,000 times the diameter of the cell where it resides. Lecture 1: Genes, genomes, gene expression etc.

28. Why study regulation of transcription ? Gene expression = Transcription + translation But it is probably fair to say that the lion share of regulation of gene expression occurs on the level of transcription.

29. Lecture 1: Genes, genomes, gene expression etc. Why study regulation of initiation of transcription? The term “transcription” includes - initiation - elongation - termination and is linked to splicing, transcript stability etc., But, again, the lion share of regulation of transcription occurs on the level of regulation of transcription initiation. Therefore, most of my lecture series is about regulation of transcription initiation.

30. Mechanistic concept of transcription: Coding strand of DNA: the strand that corresponds to the RNA used to translate the protein, running 5’ to 3’. The template strand of the DNA is transcribed to become the mRNA. 5’ 3’ 5’ 3’ DNA RNA transcription coding strand non-coding strand Lecture 1: Genes, genomes, gene expression etc.

31. What are transcription and transcription initiation? Lecture 1: Genes, genomes, gene expression etc.

32. What does 5’ end of RNA mean? Lecture 1: Genes, genomes, gene expression etc. Transcription starts with 5’ end of RNA

33. What does 5’ end of RNA mean? 5’C of terminal nucleotide (with attached triphosphate) forms end of mRNA 3’C of terminal nucleotide (and all subsequent nucleotides) becomes target of polymerization. Mechanism of polymerization: Nucleophilic attack of 3’ OH on alpha-phosphorylgroup of Incoming NTP. Lecture 1: Genes, genomes, gene expression etc. Transcription starts with 5’ end of RNA

34. Mechanism of transcription: Nucleophilic attack of 3’ OH on alpha- phosphorylgroup of incoming NTP. 5’ 3’ Lecture 1: Genes, genomes, gene expression etc.

35. Promoter:Region of DNA required for transcription initiation. Lecture 1: Genes, genomes, gene expression etc. Site of transcription initiation is called a “promoter”

36. Lecture 1: Genes, genomes, gene expression etc. Function of promoter: Nucleotide sequences recognized by RNA polymerase to bind DNA and initiate transcription. RNA polymerase is the enzyme that transcribes a gene into a mRNA. RNA polymerase

37. Lecture 1: Genes, genomes, gene expression etc. A promoter is the site where RNA polymerase binds DNA and starts to transcribe a gene. Different promoters of bacteria or of humans have very different properties. And this is what the next lectures will be about !

38. - Genes are nucleotide sequences that most often encode proteins plus the flanking regulatory regions - Transcription is the process to turn a DNA sequence into an RNA - The enzyme transcribing genes is called RNA polymerase - A promoter is the nucleotide sequence at the 5’ end of a gene required to initiate transcription - The DNA is read from the 3’ to the 5’ direction, the mRNA grows 5’ to 3’ Summary Lecture 1: Genes, genomes, gene expression etc.