This Week • Chapters 9, 10.1 and 10.4 - 10.7 for reference, – exam material will be on lecture content for the above, – sample questions, and questions for these chapters will be posted this afternoon.

• Exam Friday: Assignments in Chapters 6, 11, 8, 9 and 10. All lecture material.

Gene Expression …the processes by which information contained in genes and genomes is decoded by cells, ...in order to produce molecules that determine the phenotypes observed in organisms, – transcription (post-transcriptional modifications), – translation (post-translational modifications.

Transcription ...the synthesis of mRNA from a DNA template, - now it is important to understand when and where, as well as how.

mRNA Synthesis • Template (DNA) and

Promoter

, • Nucleoside triphosphates (NTPs), – N: A,U,G,or C, • Enzymes (

RNA polymerases

), • Energy (as in replication, from phosphate bonds).

E. coli

RNA Polymerase

RNA Polymerase Scanning

E. coli

Promoter Sequences

Promoter Regions Proximal ( ~ 100 - 200) Core Promoter ( ~ 0 to ~ -40 bp)

Regulation of Transcription (Prokaryotes) • Regulation of gene expression is often at the transcription level, – Negative regulation, • inducible, • repressible, Gene native state is “on”.

– Positive regulation Gene native state is “off”.

Protein!

Negative Regulation (Inducible) …default state is “on”, i.e., the gene is transcribed.

Protein, or metabolite, etc.

Protein!

Negative Regulation (Repressible) …default state is “on”, i.e., the gene is transcribed.

Protein, or metabolite, etc.

Positive Regulation …default state is “off”, i.e., the gene is not transcribed.

Prokaryote Review

> 8 sub-units TATA-binding protein.

TFIIA binds to the TFIID complex to block inhibitors (D-A complex).

TFIIB binds to the D-A complex.

RNA polymerase II and TFIIF complex bind.

TFIIE, TFIIH and TH FIIJ added in order.

Basal Factors

Eukaryotic Initiation

Promoter Bashing Eukaryotic Enhancers and Silencers can act at great distances.

Drosophila

Guts and Such dpp locus ...(c) and (d) are ID enhancer driven, give rise to fly appenages.

Terms •

cis

-acting elements; – DNA sequences that serve as attachments sites for the DNA binding proteins that regulate the initiation of transcription.

•

trans

-acting elements; – the DNA-binding proteins that regulate the initiation of transcription.

Chromatin Remodeling Expressed Proteins

Alternate Promoters (Figure 10.22)

Transcription Unit …the portion of a gene that specifically codes for a protein (

cistron

), plus other mRNA.

5 ’ 3 ’ transcription unit RNA polymerase begins transcription here, just “upstream” of the DNA that codes for the protein.

5’ UTR Untranslated Regions 3’ UTR UTRs are transcribed, but not translated.

UTR sequences diverge more rapidly during evolution than structural regions.

Terminator …a sequence of nucleotides (AAUAAA in the transcribed molecule) that specifies the end of the transcription unit.

Terminator 5 ’ 3 ’ RNA polymerase begins transcription here.

transcription unit

RNA Synthesis …from one strand of the double helix, DNA template strand is read 3 ’ to 5 ’ , RNA strand ‘grows from 5 ’ to 3 ’ ,

Elongation Nucleotides are added to the 3 ’ end of the elongating RNA.

mRNA vs. pre-mRNA • prokaryotic mRNA synthesis described so far requires little, or no further modification prior to translation into proteins, • eukaryotic mRNA requires extensive modifications.

Post Transcriptional Modification I 3’ UTR 5’ UTR …modified guanine cap added to the 5’ end.

…lots of adenines added to the 3’ end.

Occurs in the nucleus.

Increases stability, may help transport and sorting.

Post Transcriptional Modification II …intervening sequences (

introns

), do not code for proteins.

…code for proteins.

Introns affect expression.

Differential splicing can alter the protein’s function.

Provides “functional cassettes”, for evolutionary mixing and matching.

small nuclear Riboproteins (snRNPs) • Introns are spliced out at structure called spliceosomes, • mRNA remains relatively stable, introns are digested rapidly.

Splicosomes snRNP = small nuclear RiboNucleoProtein.

Study Figure 9.15 for Wednesday

Intron Excision

Alternate Excision (example)

Genetic Code …RNA is an intermediary in the transfer of information from DNA to the synthesis of protein, …how is that information organized?

Code is Linear The 5 ’ -3 ’ sequence from the DNA template is equivalent to the mRNA (except for uracils).

DNA code strand...

DNA template strand...

5 ’ -AAA GGC TGA TCA ATC GAT CGT GAC-3 ’ 3 ’ -TTT CCG ACT AGT TAG CTA GCA CTG-5 ’ RNA strand...

5 ’ -AAA GGC UGA UCA AUC GAU CGU GAC-3 ’

Information Capacity • need code for 20 Amino Acids, • two base-pair code, – 16 different combinations of 4 bases (4 2 ), • aa, at, ac, ag, tt, tc, tg, cc, cg, gg, ta, ca, ga, ct, gt, gc, • three base-pair code, – 64 combinations of 4 bases (4 3 ).

Codons …a triplet of nucleotide bases that specifies or encodes the information for a specific amino acid, – also need codons to indicate the beginning and end of the protein to be synthesized.

rII

Again Revertants returned the code to an “in frame” conditions.

Code is Degenerate 20 amino acid codons + start and stop codons 20-some required 64 possible All combinations are used.

Start/Stop Codons …AUG codes for the ‘start of translation’,

a methionine

, – most proteins thus begin with the amino acid

methionine

, …UAA, UAG and UGA are stop codons, indicating the C terminus of the protein.

Wobble: the third nucleotide is not necessarily specific.

Central Dogma

DNA

replication transcription

RNA

translation

Protein

Translation …the synthesis of a polypeptide. This occurs on ribosomes using the information encoded on mRNA, – tRNA molecules mediate the transfer of information between mRNA and the growing polypeptide.

tRNA

amino acid attachment site:

each amino acid has specific tRNA(s),

anti-codon:

site of interaction with the mRNA template.

The ‘Ends’ to the Means tRNA secondary structure ‘reveals’ a three base pair structure on one end, Specific anti-codons for specific amino acid designation.

“anti” = complementary

activating enzymes:

aminoacyl-tRNA synthetases, one for each amino acid.

Ribosomes …a supramolecular complex of rRNA and proteins, approximately 18 - 22 nm in diameter, …the site of protein synthesis,

Ribosome Structure

Structure/Function

E site

(exit): uncharged tRNAs are discharged from here.

P site

(peptidyl): binds the tRNA that holds the

growing polypeptide

.

A site (

amino acyl): binds the tRNA that holds the

next

amino acid,

Initiation small sub-unit and a charged Met tRNA (methionine) forms the initiation complex, large sub-unit binds the complex, P site associates with the Met tRNA.

Elongation (3 steps) (uncharged tRNA

exits)

leaves open A site, cycle repeats.

1. recognition:

tRNA anti-codon matches RNA codon, 3.

translocation

, tRNAs shift, mRNA shifts 1 codon, 2. amino acid(s) from P site tRNA transfered to the new tRNA,

peptide bond formed

,

Peptide Linkage hydrolysis reaction

Elongation (3 steps)

Peptide bond formed

.

Termination stop codon attracts a protein release factor, hydrolysis of last tRNA/amino acid yields terminal carboxyl group, ribosome disassembles.

N-Terminus --> C-Terminus ...polypeptides are synthesized beginning from the N-terminus (amino terminus) and going to the C-terminus (carboxy terminus), …this corresponds to the 5 ’ -3 ’ sequence.

DNA Coding

Wednesday