CENTRAL
DOGMA
The
Central Dogma of genetics is: DNA is transcribed to RNA which is translated to protein. Protein is never back-translated to RNA or DNA; and
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In very general terms, what does a chromosome contain?
- Information, genetic information to carry out the characteristics of life -- precise
self replication, ability to exchange energy with the environment, etc.
In very general terms, what are the two related functions of DNA?
- Information storage
- Information transfer
- DNA transcribed into RNA
- DNA's function in information transfer
What is the Central Dogma associated with information storage and retrieval?
- Central Dogma:
DNA-->RNA-->unfolded protein-->native, folded protein
What are the three processes of the central dogma?
How does DNA function as an information molecule?
- replication, DNA --> DNA
- transcription, DNA --> RNA
- translation, RNA --> unfolded protein --> folded protein
In terms of molecular conformation, what occurs through the central dogma?
- Translation of linear information, a sequence of nucleotides, into 3-D information, the
structure of a protein.
Propose a biochemical principle:
Principle of Information Transfer.
The maintenance of the high degree of structural and functional organization in an
organism is an essential prerequisite for the continuation of life. The perpetuation of
life depends on sequential and conformational molecular information.
- Concept 1: The information contained in the linear sequence of monomers in a
polymer determines higher order structure and function.
- Concept 2: The flow of biochemical information is from nucleic acid to protein,
generally DNA to RNA to Protein.
- Concept 3: Information flows from molecules in which sequencial information
predominates (nucleic acids) to molecules in which conformational information predominates
(proteins).
Central Dogma: DNA --> RNA --> unfolded protein --> folded protein
What are the differences between DNA and RNA?
- base composition: RNA = AGCU, DNA = AGCT
- carbohydrate: RNA = ribose, DNA = deoxyribose
- structure: RNA = single stranded, DNA = double helix
RNA
- usually single stranded
- linear polymer of ribonucleotides.
- Some secondary and tertiary structure but often ill-defined.
What are the different types of RNA?
What are the functions of the different types of RNA?
- messenger RNA = mRNA, information transfer
- transfer RNA = tRNA, information transfer
- ribosomal RNA = rRNA, structural
- small nuclear RNA = snRNA, ribozymes, RNA processing.
Information Transfer:
DNA replication, transcription and translation.
What is replication?
Transfer of genetic information from one generation to the next.
DNA-directed DNA synthesis: replication of the genome.
What is the structural basis for the precise duplication of the genome?
- The Watson-Crick structure of DNA: the strands are complementary, the nucleotide
sequence in one automatically specifies the other.
- The enzyme, DNA polymerase III, is very accurate: it has proof reading capabilities.
Is replication conservative or semi-conservative?
What does that mean?
- Is the parental genome of double stranded DNA fully conserved in the parental cell or is
it split equally (semi-conserved) between two daughter cells?
Replication is semi-conservative.
What is the evidence for semi-conservative replication?
Classical experiments of Meselson and Stahl. Label DNA with *heavy isotope* N15 and
allow replication in light N14: distinguish heavy, light and hybrid DNA by centrifugation.
Results: after 1 generation, each genome contains a hybrid N15-N14 DNA; after 2
generations, there are 2 hybrid and 2 light (N14-N14) genomes.
- Each strand of DNA serves as a template for the synthesis of its complement.
- The strands separate and each is used as a template for the synthesis of a
daughter strand.
- The two new double helices each contain half the parental DNA.
- This process produces a replication fork
Is replication uni-directional or bi-directional?
- Bi-directional
- Two replication forks proceeding from the origin.
What is the major replication enzyme?
DNA polymerase III, a DNA-directed DNA polymerase
- Synthesis is 5'-->3'
- Substrates are deoxynucleoside triphosphates (to make deoxyribonucleic acid)
- Proof reading , errors removed by 3'--5' exonuclease
- Processivity is very high (the ability of the enzyme to replicate a large tract of DNA
before *falling* off)
- Replication requires DNA unwinding by enzymes termed helicases: these enzymes unwind the
DNA helix before the replication fork and wind it up again afterwards.
There are large numbers of different enzymes and proteins involved at the replication
fork in the replisome.
DNA damage by UV radiation or chemicals is repaired by other DNA polymerases.
UV-damage results in adjacent T residues in one strand becoming covaletly linked to each
other, producing a thymine dimer. This causes the double helix to become distorted --
kinky.
Xeroderma pigmentosa is a genetic disorder in which patients cannot carry out
UV-radiation repair. They are very prone to skin cancer from an early age.
What is Transcription?
- Copying a gene as RNA
- DNA-directed RNA synthesis from a gene
What is a gene?
- There is no good definition of a gene!
- A sequence of DNA that is transcribed from specific start to specific stop base
sequences.
- Beadle and Tatum, working with the eukaryote mold Neurospora crassa, concluded
that one gene codes for one protein.
- But what about genes that code for RNA's like rRNA and tRNA?
- A gene is a sequence of DNA that is transcribed into a single RNA as defined by specific
start and stop sequences of bases.
- Note the circularity of the argument!
- But the single RNA may be polycistronic!
What does that mean?
- A cistron is synonymous with a gene.
- A polycistronic RNA results from the transcription of an operon.
What's an operon?
- A genetic unit containing several genes with related functions: the bacterial operon for
lactose (milk sugar) metabolism contains 3 genes coding for 3 different proteins.
- An operon is transcribed as a single unit, a polycistronic messenger RNA (mRNA) that
codes for more than one gene product.
Name 4 types of RNA.
What are their functions?
- mRNA, messenger RNA that is translated into protein
- rRNA, ribosomal RNA that, together with ribosomal proteins, forms a structural scaffold
for the translation of mRNA, the ribosome
- tRNA, transfer RNA, a specific carrier of amino acids
- snRNA, small nuclear RNA involved in processing of mRNA in the nucleus
What is the major transcription enzyme?
RNA polymerase, a DNA-directed RNA polymerase
- RNA synthesis is 5'-->3'
- substrates are ribonucleoside triphosphates ( to make ribonucleic acid)
- begins at the promoter, 5' end of the gene
- processivity is very high, proceeds to 3' end of gene without stopping or falling off
the gene
- proof reading by precise Watson-Crick base pairing, A=U and G=C
Regulation of transcription of a gene is at the 5'-end of the gene at region(s)
termed operators
- Transcription of some genes is constitutive = housekeeping genes
- Transcription of other genes is in response to a stimulus = inducible genes
What are exons and introns?
- exons are coding regions, and
- introns are non-coding regions of the mRNA transcript
- exons and introns are found in most, but not all, eukaryote genes
- introns have to be spliced out before the mRNA is translated
- splicing is by snRNA's acting as enzymes, or ribozymes, an example of the catalytic
function of RNA
Translation
- Synthesis of a linear polymer of amino acids from a linear polymer of nucleotides
Where does it occur?
On the ribosome, a rRNA-protein complex that provides:
- a scaffold for mRNA
- sites for the docking of tRNA charged with a specific amino acid
- an enzyme for peptide bond synthesis between amino acids
- an enzyme for translocation of the mRNA through the ribosome
What is the function of tRNA?
- Carrier of a specific amino acid during translation
What is the structure of tRNA?
- secondary structure has some base-pairing --> cloverleaf
- information transfer at the anti-codon loop, complementary to the codon
- note the importance of H-bonds in the genetic code
- tertiary structure is L-shaped which places the amino acid far from the codon-anticodon
site
- degeneracy of the code produces wobble
What is the genetic code?
A sequence of 3 nucleotides forms a codon
- unambiguous, each codon specifies an amino acid, or start, or stop
- degenerate, some amino acids have multiple codons
- 2-letters often sufficient, specifiy hydrophobic and hydrophillic amino acids
What is the enzyme that charges tRNA with an amino acid?
an aminoacyl-tRNA synthetase
- it has proof reading capabilities through the precise fit of amino acid and tRNA
- energy provided by ATP: energy for the formation of aminoacyl-tRNA and for proof reading
- there are at least 20 synthetases, isoaccepting for the tRNA's coding for a single amino
acid
What is the mechanism of translation?
- mRNA forms a large complex with the ribosome and protein factors
- together they guide in the correct aminoacyl-tRNA
- correct amino acid specified by codon-anticodon base pairing (H-bonds)
- protein factors have proof reading capability--energy provided by GTP
- an enzyme catalyzes polymerization of two amino acids, peptide (amide)bond formation
between two amino acids (see Chemistry lecture)
- an enzyme catalyzes movement of mRNA through the polymerization site: energy provided by
GTP
- mRNA translated from 5'--> 3', same direction as it is synthesized
Reprise:
- Flow of information: central dogma
- DNA--> RNA-->linear amino acid sequence --> 3D-conformation of protein
But some viruses have only RNA as their genome: no DNA.
How do they carry out information transfer?
How do they get around the unidirectional flow of information in the central dogma?
- Use an enzyme called reverse transcriptase to transcribe RNA into DNA.
- Example: HIV, a retrovirus
- Then, use central dogma.
- For HIV:
- RNA-->DNA--> mRNA --> linear amino acid sequence --> 3D-conformation of
protein
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