Lecture 5: Chapter 4 • Basic molecular genetic ... - Genome Tools

Lecture 5: Chapter 4 

• Basic molecular genetic mechanisms 

– Nucleic Acids 

– Information flow in biological system 

– RNA Transcription 

– Protein Translation 

– DNA replication 

– Viruses

•Classic EM DNA and RNA txn

•EM nucleolus, RNAP and rRNPs 

•“old” = heavy and radio isotopes probes 

•“new” = fluorescent and luminescent probes

Biochemistry inside the cell 

•Central Dogma

Nucleotides: Common structure

Anti vs syn

Why DNA and not RNA, if they are the same? 

•1) RNA less stable under certain conditions 

•Alkaline 

•2) C-OH more reactive, than C-H 

•3) Polymer- larger grooves, more accessible by enzymes 

•4) also, hereditary base info POV difference

Chargaff’s Rules 

•Review article, 1950” ‘Chargaff’s Rules’ - ‘regularities’ 

•Lectured at Cambridge, 1952

•1952 

•Franklin and Wilkins had a general idea what DNA looked like 

•But “They had done no proper model building”…‘missed the 

base pairing and overlooked Chargaff’s Rules’ (Crick) 

•Pauling built models but didn’t consider Chargaff’s Rules, also did not have crys data 

•http://www.dnai.org/text/mediashowcase/index2.html?id=66 

Plus…

Leads to… 

•http://nobelprize.org/educational_games/medicine/dna_double_helix/readmore.html 

•http://www.cumc.columbia.edu/news/journal/journal-o/fall-2003/dna.html 

•Nature, Apr 25, 1953 

…leading to…

‘immediate’ answers… 


•http://www.chem.ucsb.edu/~kalju/chem110L/public/tutorial/intro.html 

•http://www.dnai.org/text/mediashowcase/index2.html?id=66 

•Nature, Apr 1953

Leads to… 

•Two months earlier…. 

•Working to fit cardboard cutouts of the bases into Franklin’s data, Watson… 

•“Francis kept telling me there’s Chargaff’s pairs; would they pair to each other?” 

•“But I didn’t like Chargraff… I didn’t want to use his data in finding the structure.” 


•http://www.cumc.columbia.edu/news/journal/journal-o/fall-2003/dna.html

•dsDNA, complementary and anti-parallel

•http://www.cumc.columbia.edu/news/journal/journal-o/fall-2003/dna.html

DNA forms: A, B and Z 

•Right handed 

•Left handed

Views of the double helix

Using the double helix: Proteins ‘bridge’ 

nucleic acids to the ‘world’

Using the double helix

What is DNA? 

•Native vs denatured

Native vs denatured DNA

Using the double helix

Physicochemical characterizations

DNA secondary structures

DNA ‘bridges’ chemistry to life 

•Double strand to single strand

RNA secondary structures

Why is secondary structure, and stability of, important? 

•role in structural aspects of complexes 

•gene expression/transcription regulation 

•genome regulation (large regions)

Nucleic acids have “3-D” structure 



•http://www.dnai.org/text/mediashowcase/index2.html?id=66

Biochemistry inside the cell

Biochemistry of 

[or manipulation of] nucleic acids 

involve: 

•template 

•resolution of double-strand and 

•secondary structure 

•chemistry

ACTGGTCAACGTTGCA….. [and?]

Overview of information flow: 

Central dogma [paradigm] of molecular 

biology 

• 1958 Crick 

• Information flow 

• Described ‘knowing everything’ about 

molecular mechanisms inside cell 

• DNA DNA->RNA->protein

? 

Central dogma of molecular biology 

(but...) 

Exceptions: 

• Temin ‘64 predicted RNA viruses 

Temin, Baltimore ‘70 found RTase 

• Prusiner ~82 “prion” 

*There’s more than we know

Central dogma of molecular biology 

(but...) 

Prusiner ~82 “prion in disease” 

Prions (kuru, N. Guinea, early ‘00s; 

Gadjusek, ‘57; slow virus ‘66) 

(BSE; UK, Nov86) 

Also, 

Genome size paradox 

‘05 ‘recent’ gene estimates 

•Monod ‘54, “What was true for E. coli would be true for the elephant.” 

-> but, miRNA parallel regulatory system acting on DNA, RNA and protein 

–Allows structural complexity in humans?

Exception: CWD and 

deer 

Oct 5, 2006 EHoover 

•“Deer spread a brain-destroying disease through saliva” 

•white-tailed deer at CSU Chronic Wasting Disease Research Facility 

•saliva from CWD-infected deer to healthy tame from unaffected area 

•18 month surveillance of tonsil tissue and eventually brain 

•all deer from saliva exposure as well as single blood transfusion 

•also, additional work: not just casual contact but contact with environment, eg secretions

ED Belay et al., Emerging Infect Dis 04 

Chronic wasting disease of elk and deer in CO, WY, NE 

Spread from focus 

Food-borne transmission of BSE to humans 

Species barrier [?] 

Sept 05 WVa, MD? 

Apr 06 prions stable in soil 

Exception: Protein to protein

Genome content paradox 

[and elephants and E. coli] 

-Resolution as non-coding RNAs (siRNA, RNAi, microRNA)? 

-regulatory RNA 

2005

Central Dogma of Molecular Biology 

[seemingly ‘often refuted’ by new understandings] 

•Describes ‘information flow inside the cell’ 

•How can this be the ‘Central Dogma’ if it does not hold up? 

•LMoran blog: http://sandwalk.blogspot.com/2007/01/central-dogma-of-molecular-biology.html


[seemingly ‘often refuted’] 

•Many [younger] scientists or the Recombinant DNA, Molecular Biology and Genomics era 

read/studied Watson’s “Molecular Biology of the Gene” 

•[not many have read the original paper[s]] 

•LMoran blog: http://sandwalk.blogspot.com/2007/01/central-dogma-of-molecular-biology.html

•http://sandwalk.blogspot.com/2007/01/central-dogma-of-molecular-biology.html 


[perception corrected]

•Still, prions? 

•http://sandwalk.blogspot.com/2007/01/central-dogma-of-molecular-biology.html 


[perception corrected]

Replication: DNA chemistry 

•Biochemistry as information storage dsDNA, ssDNA

Transcription (txn): RNA chemistry 

•Biochemistry as mRNA, tRNA and rRNA, also ATP/GTP energy

Transcription (Txn) is RNA from DNA 

RNA ‘bridge’ DNA to the ‘world’

•Bacterial RNAP 

•Note DNA bend

Biochemistry of txn: Model process

Current model of RNA polymerase

•Refinements thru better 

tools, better understanding

RNA transcription: Structural basis 

RKornberg, Sci Jun01 x2 articles 

•Structural basis of transcription @2.8 and 3.3A 

•RNApol II transcribing a gene 

•Protein: gray, orange clamp and green bridge 

•DNA: blue template strd; 

•green non-template strd 

•RNA: red; 

•Mg ++ : pink

A: 

•Refined Pol II structure 

•Electron density 

•Ribbon of 10 subunits 

B: 

•Structure of Rpb1: 

•domains and domain-like regions 

R. Kornberg, Nobel Chemistry 2006 

Transcription: Structural and molecular basis

• Prokaryotes 

• Eukaryotes 

Organization of genes 

• [using the genome]

DNA to RNA to protein, what and when?

Example of “on/off:” Need tryptophan

Different strategies: Prok vs Euk

Different strategies: Euk

Eukaryotic gene structure in [a little more] detail

RNA transcript processing

Cell type-specific splicing of fibronectin 

•75 kb gene 

•Twenty isoforms, glycoprotein, ca 5% carbohydrates, where 

•EIIIB and EIIA exons encode binding domains for fibroblast surface proteins 

•Fibroblast mRNA contains both- fibronectin adheres to Extracellular Matrix 

•Hepatocyte version is missing both- secreted and circulated 

•during blood clots, fibrin-binding domains bind to fibrin; 

•complex bind integrins on platelets- cascade

Drosophila to Mammals 

•Dscam, Ig superfamily member 

•Isolated by its affinity to Dreadlocks (Dock) 

•Homolog of mammalian oncogene Nck (appearance of the photoreceptor cell axon projection pattern) 

•Dscam, Dock, Pak (Ser/Thr kinase) together direct pathfinding of 

Bolwig’s nerve, subclass of sensory axons, to target in embryo 

•Dscam required for formation of axon pathways in embryonic CNS 

•http://fruitfly.files.wordpress.com/2006/11/fruitfly.jpg 

•http://sdbonline.org/fly/hjmuller/dscam1.htm

Record alternative splicing 

•D. melanogaster Dscam gene 

•[“Down Syndrome cell adhesion molecule”] 

•Ig superfamily protein 

•115 exons of which 95 mutually exclusive exons,eg, organized into 4 clusters 

•exons 4, 6, 9 and 17 clusters contain 12, 48, 33 and 2 mutually exclusive alt exons 

•38,016 mRNA isoforms for distinct axon guidance receptors 

•for neuronal wiring specificity (innate immunity)

Good gene, bad gene [?] 

(splicing is important also as…) 

•BCL-X gene (“B-cell lymphoma”) 

•Normally, sans blue portion of Exon II, 

produces Bcl-X S mRNA, a protein that induces apoptosis 

•If splice to include all Exon II, 

produces Bcl-X L mRNA, a protein to inhibit apoptosis 

• -> Many cancers have high incidence of Bcl-X L 

• -> Successful chemotherapy results in higher proportion 

of Bcl-X S

Euk gene: “editing” post-RNA synthesis 

•http://en.wikipedia.org/wiki/Apolipoprotein_B 

•http://en.wikipedia.org/wiki/Messenger_RNA 

•Some cases,’editing’ occurs 

•Changing nucleotide composition of mRNA 

•Ex, human apolipoprotein B mRNA 

•[primary apolipoprotein of low density lipoproteins (bad)] 

•Edited in some tissues not in others 

•Two main isoforms in plasma: APOB48 and APOB100 

•First by small intestine, second by liver 

•Same single transcript >16kb 

•“48” lacks C-terminal LDL-receptor binding region 

•Editing creates early stop codon 

•FNassir… NODavidson. ‘96 JNutrition. “[ApoB] mRNA editing 

preserved in intestine and liver of Zn-deficient rats” 

•Zn: Essential trace element 

•key role in gene expression, DNA replication, cell growth and 

division; essential for many enzymes

Euk gene: polyA tails 

•http://en.wikipedia.org/wiki/Polyadenylation 

•Cleavage and polyadenylation 

•Multi-protein complex cleaves 15-30 nucleotides downstream 

•Of usually AAUAAA 

•Polyadenylate polymerase adds A+ 

•Linked to spliceosome

Euk gene: mature transcript 

•http://en.wikipedia.org/wiki/Polyadenylation

Prok gene expression regulation: Lac operon 

•catabolic pathway 

•repressor 

•inducer 

•“CAP” protein

Prok gene expression regulation: Trp operon 

• anabolic pathway 

• repressor 

• inducer 

• attenuation: 

-tRNA 

-amino acid

Finer mechanism of gene regulation at txn 

[more to learn from prokaryotes…] 

MJ Cromie et al. Cell Apr06 

•RNA sensor for intracellular Mg ++ 

•‘Mfold’ software 

•Salmonella model for regulation of Mgt Mg ++ transporter 

•Above threshold, Mg ++ binds and promotes txn stop 

•Recall Attenuation

Gene expression regulation by 

• Inducers 

• Repressors 

small molecules 

• Specific activator proteins, eg CAP

•Regulate nitrogen assimilation 

•Ex., Bradyrhizobium japonicum 

•NtcC mutants cannot use potassium nitrate 

•Lacked glnII transcripts 

•(Glutamine synthase (GS)) 

•Rice prefers ammonium over nitrate 

•Either taken up by plant or produced by 

•Reduction of nitrate, then assimilated by 

•GS produces amino group of Gln 

DNA looping

Translation: roles of RNA

Universal genetic code

Multiple reading frames 

•what does this mean?

-> mRNA is a string of codons

-> mRNA is a string of codons 

--> which encode a protein 

•http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/hbb.shtml 

•http://sickle.bwh.harvard.edu/scd_background.html 

•One base, one amino acid, one protein

•“HbA” to “HBS” 

•Red box is amino acid #6 

•G6V 

•(glutamine to valine) 

•GAG to GTG 

•http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/hbb.shtml 

•http://sickle.bwh.harvard.edu/scd_background.html 

-> mRNA is a string of codons 

-->which encode a protein 

--->that affects the whole organism, species? 

•One base, one amino acid, one protein 

•Many effects 

•Pleiotrophism

Importance of codon usage to biotechnology

Universal?

Structure of tRNA determines function 

•“translating”/decoding nucleic acid sequence 

• into amino aci sequence

tRNA

Nucleic acids have “3-D” structure 



•http://www.dnai.org/text/mediashowcase/index2.html?id=66

wobble 

•Allows tRNA to recognize >1 mRNA codon 

•Same amino acid 

•I = inosine, prs with C, A, U

RNA in translation, and proteins…

•Early attempts at structures

•E. coli 70S crystal structure 

•16S rRNA, proteins green/23S rRNA, proteins purple 

•5S rRNA dark blue

The end of the story is the beginning… 

•Termination releases all components

•After termination, re-associates to initiate 

•Preinitiation complex: 40S + eIF3 with 

•eIF1A+Met-tRNA/eIF2-GTP 

•GTPeIF2GDP 

•But eIF2-GTP only with Met-tRNA, then 

•40S and 60S subunits + eIF3, eIF6 

•Above scans for Kozak sequence: 

ACCAUGG 

Stepwise synthesis of peptide: 

Initiation of translation

elongation

Translation termination 

“function by absence”

PABP 1 and recycling translation 

•Two ways to increase protein synthesis 

•Simultaneous ribosomes and recycling 

•Circular polysomes 

•polyA-binding proteins (PABP) 

•Bridges- circular mRNA

Recycling model

Third component= DNA replication

Lecture 5: Chapter 4 • Basic molecular genetic ... - Genome Tools

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