1. Experiments demonstrated that DNA was the hereditary material
Genotype (DNA) to
Phenotype (proteins)
“Helical Man”
by David Bakalar
MBL, Woods Hole MA
How is the hereditary information read out?

2. Accuracy of DNA replication makes it a good choice for the hereditary material
1949 – Chargaff’s postulates “rules” for nucleotide bases
1869 – Meischer isolates “nuclein”
1928 – Griffith discovers heat-stable “transforming principle”
1953 – Watson and Crick construct a model for DNA structure
1900
1950
2000
1850
1910s – Levene identifies nucleotides as the building blocks of DNA
1944 – Avery et al. show DNA is the “transforming principle”
1952 – Results of “blender” experiment is consistent with DNA
Meselson and Stahl show replication is semi-conservative

3. DNA replication is FAST and PRECISE
Fast – can incorporate hundreds of nucleotides/second
DNA polymerase has some proofreading ability
Cells also have DNA repair activities
Error rate estimates range from 1/10-6 to 1/10-9
Precision of DNA replication makes it a good hereditary material

4. DNA polymerases are directional enzymes
synthesize the 5’-end of the daughter strand first
“Leading” strand is synthesized continuously in same direction as helicase
Helicase opens up helix, breaking H-bonds
“Lagging” strand is synthesized in segments, which are stitched together

5. Information transfer Genotype (DNA) to Phenotype (proteins)
“Helical Man”
by David Bakalar
MBL, Woods Hole MA
How is the language of nucleotides translated into the language of amino acids?

6. Francis Crick articulated the “central dogma” on information transfer with RNA serving as the intermediate
RNA intermediate

7. How is it different from DNA?
RNA
How is it different from DNA?
single-stranded
sugar is ribose
uracil replaces thymine
All of these changes make RNA more reactive in water – it is less stable than DNA!

8. Three classes of RNA are involved information transfer
mRNA – messenger RNA
carries sequences encoding proteins to the ribosome
tRNA – transfer RNA
carry specific amino acids to the ribosome
rRNA – ribosomal RNA
Catalyzes polymerization of amino acids into proteins
“Structural” RNAs comprise the protein synthetic machinery
Cells need many copies of these
Same tRNAs and rRNAs in all cells

9. Details of the "central dogma" were discovered using bacteria
Two processes (know the difference):
Transcription – synthesis of a RNA molecule from a DNA template – transcription is catalyzed by RNA polymerase
Translation – synthesis of protein using codons in mRNA as a template – translation is catalyzed by the ribosome

10. Bacteria (prokaryotes) lack nuclei
Translation begins before transcription is complete
Single circular chromosome (light area)
Cytoplasm is filled with ribosomes
Experiments that elucidated information transfer were carried out in bacteria

11. The DNA coding sequence is copied into RNA by an RNA polymerase
Transcription
The DNA coding sequence is copied into RNA by an RNA polymerase
template strand
coding strand
Transcript
RNA has the same sequence as the coding strand, except that uracil (U) replaces thymine (T)

12. How does RNA polymerase know where and when to transcribe RNA?
Cell-specific transcription factors open up the DNA at the promoter, allowing RNA polymerase to bind
Promoters contain binding sites for RNA polymerases

13. Translation
Nucleotide sequence is translated into a protein sequence at the ribosome
"Waltz of the polypeptide"

14. Proteins are polymers of amino acids linked by peptide bonds
Proteins fold into a remarkable array of 3-dimensional shapes determined by the sequence of R groups, each of which has a different chemistry

15. Translation uses “structural” RNAs
RNA is able to fold into a variety of conformations
Modern ribosome is an RNA catalyst (ribozyme)
RNA world? Was RNA the original hereditary material?

16. The translator: transfer RNA (tRNA)
small RNAs
75-80 nucleotides
Crystal structure of charged tRNA

17. 4 different nucleotides are found in RNAs
How does the code work?
4 different nucleotides are found in RNAs
20 different amino acids are found in proteins

18. Triplet of nucleotides (codon) in mRNA specifies an amino acid
tRNAs have anticodons complementary to codons in mRNA

19. punctuation marks: three stop codons do not code for amino acids

20. The fabricators: ribosomes
Ribosomes have 2 subunits
Each subunit is composed of a large number of proteins and 1-3 RNA molecules (1 RNA molecule is VERY long)
The RNA portion of the ribosome actually catalyzes protein synthesis
Crystal structure of large ribosomal subunit
each protein is shown with a different color
rRNA is shown in red
PDB 1ffk

21. Ribosomes are composed of a large and a small subunit
Each subunit contains a small number of RNAs and many proteins
ribosomes are large enough to be visible in electron micrographs

22. Electron micrograph reconstruction (NOT A MOVIE!)
Ribosomes rapidly associate with mRNAs immediately after they leave the nucleus, initiating translation
large mRNA passing through nuclear pore
Multiple ribosomes bind to mRNA and begin translation
Translation continues in cytoplasm

23. tRNA with amino acid binds here
tRNA with peptide chain binds here
tRNA is ejected here

24. Passing the baton: tRNAs twist, bringing the peptide chain and amino acid into close contact, resulting in a peptide bond
Growing peptide chain bound to tRNA in P site is transferred to amino acid attached to tRNA in the A site

25. RNA acts as the intermediary between DNA (the information) and protein (catalysts and structural units in cells)
RNA differs from DNA in that it is single-stranded, contains uracil in the place of thymine and ribose sugar in the place of deoxyribose.
Three kinds of RNA are found in cells: messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA)
Transcription refers to the synthesis of RNA from a DNA template
Genes contain regulatory sequences in addition to the coding sequences for proteins – transcription factors bind to regulatory sequences
RNA polymerases catalyze the synthesis of RNA molecules
RNA polymerases bind to promoter sequences and move processively along the DNA templates as they synthesize RNA molecules
Translation refers to the synthesis of protein
Ribosomal RNA forms the scaffold for ribosomes and is responsible for catalyzing protein synthesis
Transfer RNAs carry activated amino acids to the ribosomes.
Anticodon sequences on tRNAs bind codons in mRNAs.