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

Bio99A: Molecular Biology, Spring 2010

Part 2: Gene expression / transcription

Hans-Ulrich Bernard (Uli Bernard)

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: [email protected] (but I cannot answer 400 emails per week)

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

I worked for 15 years at the National University of Singapore,where I gave similarlectures as this one,

but this is my firstBio99 lecture at UCI.

This means: I know the science,but I am still learning about the student population.

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.

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

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

Lecture 1:

Genes, genomes, gene expression,What is transcription initiation?

Lecture 1: Genes, genomes, gene expression etc.

What is “gene expression”?

Central concept “dogma” of molecular biology:


Transcription + translation leads from storage form of genetic information (DNA) to functional proteins.

What is “gene expression”?


Transcription + translation leads from storage form of genetic information (DNA) to functional proteins.

This lecture series is about transcription.

Terminology: What is a gene?(simple model, appropriate for prokaryotes)


Coding sequences plus flanking elements involved in expression.

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 regiondetermines transcription terminationcan contain regulatory elements

5’ flanking region= 5’ non-coding region= upstream regioncontains regulatory elements

mRNA

Transcription + splicing

These segments have to be identified by sequence analysesand in functional studies, to detect meaning in a seeminglyfeatureless DNA double helix.


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 sequenceslike rRNA and tRNA

What is an operon? A set of genes, 2 or more, that are regulated and expressed togetheron a single mRNA (polycistronic mRNA). The genes in an operonhave related functions.


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”).


What is a genome ?

Genome size in nucleotide pairs for various organisms:

about 4000 genes

about 30,000 genes


Sum of all genes plus non-coding sequences !

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



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



Irrespective of pro- or eukaryote, genes can be on either strand of a double-stranded DNA

Transcript map of protein coding transcripts in Adenovirus







Prokaryotic genomes are very compact:

- very little space between genes - very little unfunctional (“junk”) DNA- existence of “operons”


Eukaryotic genomes are not compact



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


(= histones, chromatin) - genome located in nucleus, transcription occurs

in nucleus, translation in cytoplasm







Localization of genome and gene expression in prokaryotes versus eukaryotes


Why study regulation of gene expression?


Bacterium: 4000 + genesHigher organism: 30,000 genes

At any given time only a subset of these genes is expressed.

And this subset determines:

physiological propertiesmorphological propertiesdifferentiation, health and disease,etc. etc.

Gene expression:

Be aware of the dimensions of the undertaking:

1014 cells, each with the same30.000 genes

regulated gene expression

Perfect human being


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.



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.


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 transcriptionoccurs on the level of regulation of transcription initiation.

Therefore, most of my lecture series is about regulation of transcription initiation.

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’

5’3’

3’5’

3’

DNA

RNA

transcription

coding strand

coding strand

non-coding strand


What are transcription and transcription initiation?


What does 5’ end of RNA mean?


Transcription starts with 5’ end of RNA

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 ofIncoming NTP.


Transcription starts with 5’ end of RNA

Mechanism of transcription:Nucleophilic attack of 3’ OH on alpha-phosphorylgroup of incoming NTP.

5’

5’

3’

3’


+1 +10 +20-30 -20 -10-40

“Upstream” (5’ Flank)

“Downstream”

mRNA “start site”

3’5’

mRNA

5’

3’

“Promoter”

“Promoter”=Region of DNA required for RNA polymerase binding and transcription initiation

+1

TTGACA

“-35”

TATAAT

“-10”

16-19 bases 5-10 bases

+1

GENERAL PROMOTER STRUCTURE; -35 AND -10 HOMOLOGY REGIONS (SIMILAR IN ALL PROMOTERS)

T82T84G78A65C54A45 T80A95T45A65A50T96

Promoter: Region of DNA required for transcription initiation.


Site of transcription initiation is called a “promoter”

+1 +10 +20-30 -20 -10-40

“Upstream” (5’ Flank)

“Downstream”

mRNA “start site”

3’5’

mRNA

5’

3’

“Promoter”

“Promoter”=Region of DNA required for RNA polymerase binding and transcription initiation

+1

TTGACA

“-35”

TATAAT

“-10”

16-19 bases 5-10 bases

+1

GENERAL PROMOTER STRUCTURE; -35 AND -10 HOMOLOGY REGIONS (SIMILAR IN ALL PROMOTERS)

T82T84G78A65C54A45 T80A95T45A65A50T96


Function of promoter: Nucleotide sequences recognized by RNA polymerase to bind DNAand initiate transcription.

RNA polymerase is the enzyme that transcribes a gene into a mRNA.

RNA polymerase


A promoter is the site where RNA polymerase binds DNAand starts to transcribe a gene.

Different promoters ofbacteriaor of humanshave very different properties.

And this is what the next lectureswill be about !

- 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


Documents

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