DNA ...the genetic material.

Deoxyribonucleic Acid:

– a double stranded, helical nucleic acid molecule capable of replicating and determining the inherited structure of a cell’s proteins.

Understand how DNA and the information it contains is…

- replicated,

- expressed,

- altered by mutation.

Goals

Central Dogma(s)

DNA

RNA

transcription

Protein

translation

Genome

transcription

Transcriptome

Proteome

translation

Replication

• Genome... the dynamic complement of heritable genetic material,

• Transcriptome... mRNA component in an individual,

– complexity increases resulting from transcription control and transcription and post-

transcription modifications,

• Proteome... the protein component of an individual,

– complexity increases due to translational efficiancy, post-translational modification,

protein-protein interactions, etc.

Expanded Central Dogma

Genome Transcriptome Proteome

Complexity

• 1021 stars (estimated) in the Universe,

– astronomical,

• If the human genome codes for ~100,000 proteins, all possible combinations of genes being off or on yields ~1030,000 discrete states,

– per cell,– at any one moment over the span of a lifetime.

But, only ~20,000 genes?

Central Dogmagenetics focus

DNA

RNA

Protein

transcription

translation

Replication

Nucleic AcidsDNA/RNA

…polymers consisting of monomers termed nucleotides, A, G, C, T/U

• nucleotides: a molecule composed of,

– a pentose sugar,– a phosphate group,– and an organic molecule called a nitrogenous base.

Phosphodiester Bonds

Note 5’- 3’Orientation

5’

3’

PolynucleotidesPhosphodiester BackbonesMb long “Bases”

AdenineThymineGuanineCytosine

Phosphodiester Backbones

Bases with hydrogen bonds.

Two Hydrogen Bonds Three Hydrogen Bonds

Base Pairing A -- Tcomplementary

Base Pairing G -- Ccomplementary

Anti-Parallel

...DNA is a double stranded molecule and orients in an anti-parallel fashion,

…orientation is in reference to the phosphodiester bonds.

Complementary: strands aren’t identical, yet…????????

Please study this slide.

Complementary StrandsTemplates for Copying

…single-stranded DNA can serve as a template for high-fidelity “duplication”,

– makes DNA for growth, repair and hereditary purposes (Replication),

– makes RNA for the synthesis of proteins (Transcription).

Strands can be separated.

DNA Replicationmeiosis I

5’-AAAGGCTGATCA-3’

3’-TTTCCGACTAGT-5’

3’-TTTCCGACTAGT-5’5’-AAAGGCTGATCA-3’

3’-TTTCCGACTAGT-5’

5’-AAAGGCTGATCA-3’

A- a-

B- b-

-A -a

-b-B

Homologous Chromosomes

One Chromosome

Sister Chromatids

DNA Replication

• Template,

• Enzymes,

• Primer (to prime synthesis),

• dNTPs:

– d (deoxy), N (A,T,G,or C),

• Energy.

Helicases: enzymes that unwind the DNA double helix for DNA replication…

– Dna/A, Dna/B, Dna/C,

- proteins that bind to specific DNA sequences.

Enzyme #1

Enzyme 2

…Primase: provides a short, complementary strand of RNA that is required for DNA synthesis from a naked DNA template.

Primase

DNA polymerase III: the enzyme that adds complementary nucleotides to the backbone, based on the sequence of the single stranded template.

Enzyme 3

5’ --> 3’ synthesis.Energy

DNA Synthesistest yourself

Fidelity So FarMitosis

…the initial synthesis produces errors at a rate of 1 in 105-8,

…proof-reading during replication improves the error rate to 1 in 108-12,

…this occurs at a rate of up to 1000 bp a second.

Central Dogmagenetics focus

DNA

RNA

Protein

transcription

translation

RNA Structure

…single stranded,

…can form base pairing with DNA, or RNA,

…no simple regular secondary structure.

mRNA, tRNA and rRNA

…mRNA (messenger RNA): a type of RNA synthesized from DNA that specifies the primary structure for a protein,

…tRNA (transfer RNA): an RNA molecule that acts as an ‘interpreter’ between nucleic language and protein language by picking up specific amino acids and recognizing the appropriate codons in the mRNA,

…rRNA (ribosomal RNA): together with proteins, it forms the structure of ribosomes that coordinate the sequential coupling of tRNA molecules to the series of mRNA codons.

…non-transcribed DNA, a short distance from 5’end of a gene,

…RNA polymerase is weakly attracted to DNA in general, but is strongly attracted to promoter sequences and associated molecules.

Structural Region(peptide sequence)

5’ 3’

Promoter

Gene Expression …refers to cellular control of transcription,

5’ DNA sequences and associated molecules …

– direct when and where a gene is expressed,

– influence the amount of expression,

• strong promoter (high rate of transcription),

• weak promoter (low rate of transcription).

E. coli Promoter Sequencesconsensus sequence alignment (from a MSA)

Multiple Sequence Alignment

Promoter Regions

DNA sequence changes alter promoter function.

Transcription Factor 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.

Modulators of expression can act at great distances.

Eukaryotic Initiation

Apply the terms from the previous slide to the appropriate components on this figure.

Please study this slide.

Structural Regioncoding region

5’ 3’

transcription unit

RNA polymerase begins transcription here.

Terminator

…the portion of a gene that specifically codes for a protein.

Complementary StrandsTemplates for Copying

…single-stranded DNA can serve as a template for high-fidelity “duplication”,

– makes DNA for growth, repair and hereditary purposes (Replication),

– makes RNA for the synthesis of proteins (Transcription).

Strands can be separated.

RNA Synthesis

DNA template strand is read 3’ to 5’,

…from one strand of the double helix,

RNA strand ‘grows from 5’ to 3’,

Elongation

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

Coding strand

• Template (DNA) and Promoter,

• Nucleoside triphosphates (NTPs),

–N: A,U,G,or C,

• Enzymes (RNA polymerases),

• Energy (phosphate bonds).

Question: what does the coding strand sequence tell you about the mRNA sequence?

hnRNA vs. mRNAheterogeneous nuclear vs. messenger

• prokaryotic mRNA synthesis described so far requires little, or no further modification prior to translation into proteins,

• eukaryotic transcripts requires extensive modifications.

Post Transcriptional Modification Ieukaryotes

Occurs in the nucleus. Increases stability, may help transport and sorting.

…modified guanine cap added to the 5’ end.…lots of adenines added to the 3’ end.

Post Transcriptional Modification II eukaryotes

Introns may alter expression. Differential splicing can alter the final protein’s structure and function.Provides “functional cassettes”, for evolutionary mixing and matching.

Intervening sequences (introns), do not code for proteins.

Exons code for peptides.

Eukaryotic Intron Excision(sequence is important)

Spliceosomesconfer sequence specificity

... small nuclear RNAs (snRNAs):

– RNA molecules that act as catalysts in spliceosomes,

…work in concert with > 100 proteins to facilitate intron identification and removal,

– snRNPs: RNA/Protein structures.

U1 and U2snRNAs

U1 binds to the 5’ exon/intron junction.

U2 binds to the adenosine region at the branch site.

Psuedouradine (

Think about the required specificity for intron identification in cells.

Alternate mRNA Procesing

…alternate splicing.

Polyadenylation…lots of adenines.

AAUAA: consensus poly-A recognition site.

…complicated biochemistry, lots of protein sub-units,

- endonuclease,

- polyadenylate polymerase,

Alternate mRNA Processing

…recognition of different poly-A sites. …alternate splicing.

~20,000 genes --> ~100,000 proteins

Complexity Calcitonin gene

GeneticsIn the News

“Devil Anse”Hatfield

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

Translation

…RNA is an intermediary in the transfer of information from DNA to the synthesis of protein,

…how is that information organized?

mRNA, tRNA and rRNA

…mRNA (messenger RNA): a type of RNA synthesized from DNA that specifies the primary structure for a protein,

…tRNA (transfer RNA): an RNA molecule that acts as an ‘interpreter’ between nucleic language and protein language by picking up specific amino acids by recognizing the appropriate codon in the mRNA,

…rRNA (ribosomal RNA): together with proteins, it forms the structure of ribosomes that coordinate the sequential coupling of tRNA molecules from the series of mRNA codons.

…involved in translation.

The ‘Ends’ to the Means

Specific anti-codons for specific amino acids.“anti” = complementary

tRNA secondary structure ‘reveals’ a three base pair structure on one end,

Ribosomes

…a supramolecular complex of rRNA and proteins, approximately 18 - 22 nm in diameter,

…the site of protein synthesis,

Central Dogmagenetics focus

DNA

RNA

Protein

transcription

translation

Elongation (3 steps)

1. recognition: tRNA anti-codon complements RNA codon,

2. amino acid(s) from P site tRNA transfered to the new tRNA, peptide bond formed,

3. translocation, tRNAs shift,

mRNA shifts 1 codon,

leaves open A site, cycle repeats.

(uncharged tRNA exits)

Peptide Linkage

How does N-terminus and C-terminus relate to DNA 5’, 3’ orientation?

Peptide Sequence 1o, 2o, 3o, 4o Structure/Function

What’s 5’, 3’, N-terminus, C-terminus?

How does a base change, change things?…if you understand this, you’ve got it.

in the promoter region,

in an exon,

in an intron,

at a splice site,

at an AAUAAA site,

etc.

5’ 3’

3’ 5’

5’

5’

3’5’

N- -C

enhancer, silencer, core promoter?

? ? ? ??

AAUAAA

For Monday

Reading assignment:

Chapter 2

Review Chapter 6, and/or your General Biology Text for

Central Dogma