How to Study DNA 1.Genetic material 2.Expression product

What is gene expression? The activation of a gene that results in a protein. The activation of a gene that results in a protein. Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product. A gene is expressed when its biological product is present and active. Gene expression is regulated at multiple levels.

Expression of Genetic Information Production of proteins requires two steps: Transcription involves an enzyme (RNA polymerase) making an RNA copy of part of one DNA strand. Transcription involves an enzyme (RNA polymerase) making an RNA copy of part of one DNA strand. There are four main classes of RNA: i. Messenger RNAs (mRNA), which specify the amino acid sequence of a protein by using codons of the genetic code. ii. Transfer RNAs (tRNA). iii. Ribosomal RNAs (rRNA). iv. Small nuclear RNAs (snRNA), found only in eukaryotes. Translation converts the information in mRNA into the amino acid sequence of a protein using ribosomes, large complexes of rRNAs and proteins. Translation converts the information in mRNA into the amino acid sequence of a protein using ribosomes, large complexes of rRNAs and proteins.

Expression of Genetic Information Only some of the genes in a cell are active at any given time, and activity also varies by tissue type and developmental stage. Only some of the genes in a cell are active at any given time, and activity also varies by tissue type and developmental stage. Regulation of gene expression is not completely understood, but it has been shown to involve an array of controlling signals. Regulation of gene expression is not completely understood, but it has been shown to involve an array of controlling signals. a. Jacob and Monod (1961) proposed the operon model to explain prokaryotic gene regulation, showing that a genetic switch is used to control production of the enzymes needed to metabolize lactose. Similar systems control many genes in bacteria and their viruses. b. Genetic switches used in eukaryotes are different and more complex, with much remaining to be learned about their function.

Steps of gene expression Transcription – DNA is read to make a mRNA in the nucleus of our cells Transcription – DNA is read to make a mRNA in the nucleus of our cells Translation – Reading the mRNA to make a protein in the cytoplasm Translation – Reading the mRNA to make a protein in the cytoplasm

Structural genes: DNA that code for a specific polypeptide (protein) Promoter : DNA segment that recognizes RNA polymerase Operator : Element that serves as a binding site for an inhibitor protein (modulator) that controls transcription Three (3) regulatory elements of transcription

7 Promoter Region on DNA Upstream from transcription start site Initial binding site of RNA polymerase and initiation factors (IFs) Promoter recognition: a prerequisite for initiation Prokaryotic promoter regions -10 site: “TATA” box -35 site = TTGACA

Promoter Region on DNA

Pol II Eukaryotic Promoter Elements GC box ~200 bp CCAAT box ~100 bp TATA box ~30 bp Gene Transcription start site (TSS) Exon Intron Exon

Pol II Eukaryotic Promoter Elements Cap Region/Signal n C A G T n G TATA box (~ 25 bp upstream) T A T A A A n G C C C CCAAT box (~100 bp upstream) T A G C C A A T G GC box (~200 bp upstream) A T A G G C G nGA

General modulators of transcription Modulators: Modulators: (1) specificity factors, (2) repressors, (3) activators 1.Specificity factors: Alter the specificity of RNA polymerase s 70 s 32 Heat shock geneHousekeeping gene Heat shock promoter Standard promoter

Modulators of transcription Repressors: mediate negative gene regulation may impede access of RNA polymerase to the promoter actively block transcription bind to specific “operator” sequences (repressor binding sites) Repressor binding is modulated by specific effectors Coding sequence Repressor Operator Promoter Effector (e.g. endproduct)

Negative regulation Repressor Effector Example: lac operon RESULT: Transcription occurs when the gene is derepressed

Negative regulation Repressor Effector (= co-repressor) Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism

Modulators of transcription 3. Activators: mediate positive gene regulation bind to specific regulatory DNA sequences (e.g. enhancers) enhance the RNA polymerase -promoter interaction and actively stimulate transcription common in eukaryotes Coding sequence Activator promoter RNA pol.

Positive regulation RNA polymerase Activator

Positive regulation RNA polymerase Activator Effector

Prokaryotic gene organization Prokaryotic transcriptional regulatory regions (promoters and operators) lie close to the transcription start site Functionally related genes are frequently located near each other These “operons” are transcribed into a single mRNA with internal translation initiation sites

Prokaryotic Gene Expression PromoterCistron1Cistron2CistronNTerminator TranscriptionRNA Polymerase mRNA 5’3’ Translation Ribosome, tRNAs, Protein Factors 12N Polypeptides N C N C N C 123 Expression mainly by controlling transcription

Operons Genes that work together are located together A promoter plus a set of adjacent genes whose gene products function together. They are controlled as a unit They usually contain 2 –6 genes (up to 20 genes) These genes are transcribed as a polycistronic transcript. It is relatively common in prokaryotes It is rare in eukaryotes

Operon System

The lactose (lac) operon Contains several elementsContains several elements –lacZ gene = β-galactosidase –lacY gene = galactosidase permease –lacA gene = thiogalactoside transacetylase –lacI gene = lac repressor –P i = promoter for the lacI gene –P = promoter for lac-operon –Q 1 = main operator –Q 2 and Q 3 = secondary operator sites (pseudo-operators ) PiP ZYA I Q3 Q1 Q2

Regulation of the lac operon PiP ZYA I Q3 Q1 Q2 Inducer molecules→ Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside) - synthetic inducer, not metabolized lacI repressor PiP ZYA I Q3 Q1 Q2 LacZLacYLacA

The lac operon: model for gene expression Includes three protein synthesis coding region-- sometimes called "genes" as well as region of chromosome that controls transcription of genes Genes for proteins involved in the catabolism or breakdown of lactose When lactose is absent, no transcription of gene since no need for these proteins When lactose is present, transcription of genes takes place so proteins are available to catalyze breakdown of lactose

Eukaryotic gene

Eukaryotic gene Expression 1.Transcripts begin and end beyond the coding region 2.The primary transcript is processed by: 5’ capping 3’ formation / polyA splicing 3.Mature transcripts are transported to the cytoplasm for translation

Regulation of gene expression Plasmid Gene (red) with an intron (green)Promoter 2. Transcription Primary transcript 1. DNA replication 3. Posttranscriptional processing 4. Translation mRNA degradation Mature mRNA 5. Posttranslational processing Protein degradation inactive protein active protein single copy vs. multicopy plasmids

Regulation of gene expression Gene expression is regulated—not all genes are constantly active and having their protein produced Gene expression is regulated—not all genes are constantly active and having their protein produced The regulation or feedback on gene expression is how the cell’s metabolism is controlled. The regulation or feedback on gene expression is how the cell’s metabolism is controlled. This regulation can happen in different ways: This regulation can happen in different ways: 1. Transcriptional control (in nucleus): e.g. chromatin density and transcription factors 2. Posttranscriptional control (nucleus) e.g. mRNA processing 3. Translational control (cytoplasm) e.g. Differential ability of mRNA to bind ribosomes 4. Posttranslational control (cytoplasm) e.g. changes to the protein to make it functional When regulation of gene expression goes wrong—cancer! When regulation of gene expression goes wrong—cancer!

Transcription

Eukaryotic gene expression

Gene regulation of the transcription Chr. I Chr. II Chr. III Condition 1 “turned on” “turned off” Condition 2 “turned off” “turned on” constitutively expressed gene induced gene repressed gene inducible/ repressible genes

Gene regulation constitutively expressed gene Condition 3 Condition 4 upregulated gene expression down regulated gene expression

Definitions Constitutively expressed genes Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells. Inducible genes Genes that are transcribed and translated at higher levels in response to an inducing factor Repressible genes Genes whose transcription and translation decreases in response to a repressing signal Housekeeping genes – –genes for enzymes of central metabolic pathways (e.g. TCA cycle) – –these genes are constitutively expressed – –the level of gene expression may vary

Post-Transcriptional Modification in Eukaryotes Primary transcript formed first Primary transcript formed first Then processed (3 steps) to form mature mRNA Then processed (3 steps) to form mature mRNA Then transported to cytoplasm Then transported to cytoplasm Step 1: 7- methyl-guanosine “5’-cap” added to 5’ end Step 2: introns spliced out; exons link up Step 3: Poly-A tail added to 3’ end mature mRNA 5’-cap- exons -3’ PolyA tail

36 Intron Splicing in Eukaryotes Exons : coding regionsExons : coding regions Introns : noncoding regionsIntrons : noncoding regions Introns are removed by “splicing”Introns are removed by “splicing” AG at 3’ end of intron GU at 5’ end of intron

37 Splicesomes Roles in Splicing out Intron RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomes

38 5’ exon then moves to the 3’ splice acceptor site where a second cut is made by the spliceosome 5’ exon then moves to the 3’ splice acceptor site where a second cut is made by the spliceosome Exon termini are joined and sealed Exon termini are joined and sealed Splicesomes Roles in Splicing out Intron

Translation Three parts: 1.Initiation: start codon (AUG) 2.Elongation: 3.Termination: stop codon (UAG)

Translation P Site A Site Large subunit Small subunitmRNA AUGCUACUUCG

Initiation mRNA AUGCUACUUCG 2-tRNA G aa2 AU A 1-tRNA UAC aa1 anticodon hydrogen bonds codon

mRNA AUGCUACUUCG 1-tRNA2-tRNA UACG aa1 aa2 AU A anticodon hydrogen bonds codon peptide bond 3-tRNA GAA aa3

mRNA AUGCUACUUCG 1-tRNA 2-tRNA UAC G aa1 aa2 AU A peptide bond 3-tRNA GAA aa3 Ribosomes move over one codon (leaves)

mRNA AUGCUACUUCG 2-tRNA G aa1 aa2 AU A peptide bonds 3-tRNA GAA aa3 4-tRNA GCU aa4 ACU

mRNA AUGCUACUUCG 2-tRNA G aa1 aa2 AU A peptide bonds 3-tRNA GAA aa3 4-tRNA GCU aa4 ACU (leaves) Ribosomes move over one codon

mRNA GCUACUUCG aa1 aa2 A peptide bonds 3-tRNA GAA aa3 4-tRNA GCU aa4 ACU UGA 5-tRNA aa5

mRNA GCUACUUCG aa1 aa2 A peptide bonds 3-tRNA GAA aa3 4-tRNA GCU aa4 ACU UGA 5-tRNA aa5 Ribosomes move over one codon

mRNA ACAUGU aa1 aa2 U primary structure of a protein aa3 200-tRNA aa4 UAG aa5 CU aa200 aa199 terminator or stop or stop codon codon Termination

P SiteA Site E Site Amino Acids forming Peptide chain Ribosome tRNA anti-codon codon Translation UAC AUG Tyr GUA CAU Val mRNA strand 3’ 5’ HisMet Pro GGA CCU

Translation The difference Eukaryotic and prokaryotic translation can react differently to certain antibioticsEukaryotic and prokaryotic translation can react differently to certain antibiotics  Puromycin an analog tRNA and a general inhibitor of protein synthesis  Cycloheximide only inhibits protein synthesis by eukaryotic ribosomes  Chloramphenicol, Tetracycline, Streptomycin inhibit protein synthesis by prokaryotic ribosome

End Product The end products of protein synthesis is a primary structure of a protein. The end products of protein synthesis is a primary structure of a protein. A sequence of amino acid bonded together by peptide bonds. A sequence of amino acid bonded together by peptide bonds. aa1 aa2 aa3 aa4 aa5 aa200 aa199

Polyribosome Groups of ribosomes reading same mRNA simultaneously producing many proteins (polypeptides).Groups of ribosomes reading same mRNA simultaneously producing many proteins (polypeptides). incoming large subunit incoming small subunit polypeptide mRNA

Prokaryotes vs eukaryotes: key points Prokaryotes Eukaryotes Polycistronic mRNAs (single mRNA, multiple ORFs) Monocistronic RNAs (One mRNA, one protein) Operons (functional grouping) No splicing Ribosome scanning Often spliced Regulatory sequences lie near (~100 bp) the start site Regulatory sequences can be far (>1 kb) from the start site Translation is concurrent with transcription RNA processing is concurrent with transcription; translation occurs in a separate compartment

TYPES OF PROTEINS Enzymes (Helicase) Enzymes (Helicase) Carrier (Haemoglobine) Carrier (Haemoglobine) Immunoglobulin (Antibodies) Immunoglobulin (Antibodies) Hormones (Steroids) Hormones (Steroids) Structural (Muscle) Structural (Muscle) Ionic (K+,Na+) Ionic (K+,Na+)

Coupled transcription and translation in bacteria

VALINE HISTIDINE LEUCINE PROLINETHREONINE GLUTAMATE VALINE original base triplet in a DNA strand As DNA is replicated, proofreading enzymes detect the mistake and make a substitution for it: a base substitution within the triplet (red) One DNA molecule carries the original, unmutated sequence The other DNA molecule carries a gene mutation POSSIBLE OUTCOMES: OR

A summary of transcription and translation in a eukaryotic cell A summary of transcription and translation in a eukaryotic cell