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
- DNA replication
- 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.
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.
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 nucleotide
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
- 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.