Big Questions

• How is the structure of DNA related to its function?

• How does DNA allow for heritability?

• How does DNA allow for traits in an organism?

• How do mutations affect DNA structure and function?

Central Dogma

• "Central Dogma": Term coined by Francis Crick to explain how information flows in cells.

Replication

• Replication can only begin at specific locations ("origins") on a chromosome

• Once it begins, replication proceeds in two directions from the origin– The replication bubble

1. Helicase: Opens the helix (which causes strand separation)

2. Single Strand Binding Proteins ("SSBP's"): Keep the strand open

3. Primase: Puts down a small RNA primer which is necessary for DNA polymerase to bind to at the origin.

4. DNA Polymerase: Enzyme Responsible for DNA synthesis.

5. Topoisomerase: Rotates the DNA to decrease torque (which would shred the helix.

• Nucleotides are added to the 3' end of the DNA strand

• DNA replication can only occur in the 5' to 3' direction

• DNA is "anti-parallel": Both strands have opposite 5' to 3' orientations (one is "upside-down" compared to the other)

2. Elongation

• Nucleotides are added to the new strand of DNA in the 5' to 3' direction.

• There is an issue: DNA is anti-parallel

• As the replication machinery moves along the chromosome, only one strand of DNA (the "leading strand") can be made in a continuous, 5' to 3' piece.

• The other strand (the "lagging strand") has to be made in smaller, discontinuous 5' to 3' segments ("Okazaki fragments") which are then stitched together by the enzyme ligase

Joining• DNA polymerase removes RNA primer and fills

with DNA nucleotide• DNA Ligase – links two sections of DNA together

Replication Fork

3. Termination

• Elongation continues until replication bubbles merge

• The ends of linear eukaryotic chromosomes pose a unique challenge

• Each round of replication shortens the 5' end of the lagging strand (by about 100-200 bp)

• If this continued indefinitely, chromosomes would get shorter and shorter after each replication. – Information would start to be lost

Telomeres

• Ends of eukaryotic chromosomes• short, repeating DNA sequence

– Vertebrate Telomere: TTAGGG• TELOMERASE - enzyme responsible for

replicating the ends of eukaryotic chromosomes

• Uses an RNA template to add more telomere sequence during replication

Proofreading• There are 5 different DNA polymerases described

in prokaryotic cells. – serve a variety of functions

• DNA polymerase III- Is responsible for elongation– Rate of elongation is ~500 bases/second in E. coli– The eukaryotic analog DNA polymerase elongates at a

rate of ~50 bases/second• The initial error rate is 1 in 10,000

– 300,000 mutations every time a human cell divided• Proof-reading reduces error rate to 1 in 10 billion

(less than 1 per 3 human cell divisions)

Transcription

1. Initiation• RNA polymerase attaches to a "promoter"

region in front ("upstream") of a gene• Promoters have characteristic DNA sequences

(ex "TATA Box" in eukaryotes)

2. Elongation

• Similar to DNA replication, RNA production occurs in a 5' to 3' direction.

• The template strand of DNA is the one that the RNA transcript is being produced off of (sequence is opposite to the transcript)

3. Termination

• Transcript production continues until the end of the transcription unit is reached.

Types of RNA

• Messenger RNA (mRNA): complementary to DNA – C=G, A=U– Travel from nucleus to ribosome– Direct synthesis of protein

• Transfer RNA (tRNA): brings amino acids to ribosome

• Ribosomal RNA (rRNA): Major structural building block of ribosomes

Transcription happens in the nucleus. An RNA copy of a gene is made.

Then the mRNA that has been made moves out of the nucleus into the cytoplasm

Once in the cytoplasm, the mRNA is used to make a protein

Cytoplasm of cell

Nucleus

DNAmRNA

mRNA

Processing mRNA

• A modified nucleotide is added to the 5' end of the transcript.

• A tail of several hundred adenine residues is put on the 3' end of the transcript.

• These modifications function in nuclear export and maintenance of the mRNA

Exon Splicing

• Eukaryotic genes contain large stretches of non-coding DNA ("introns") interspersed between coding DNA ("exons")

• To produce a functional protein, the introns must be removed– the exons must be spliced together prior to the

movement of the mRNA transcript to the nucleus

• This process is accomplished by a spliceosome

Why Introns?

• Not really answered.

• Evolutionary baggage? Selfish genes?

• We do know that having multiple exons in a gene allows eukaryotes to make multiple functional proteins from one gene ("alternative splicing")

Translation

RNA polypeptide

Ribosomes– 2 subunits – only together during translation– Attaches to mRNA strand

tRNA• 3 base sequence at the bottom – anticodon

– Matches the codon on mRNA strand

The Ribosome

• The site of protein synthesis• All cells have ribosomes• Composed of two subunits• Has three "sites":

– A site: "Aminoacyl"- amino acids enter ribosome– P site: "peptidyl"- growing polypeptide is kept– E site: "exit"- empty tRNA molecules leave

tRNA• Transfer RNA molecules• Brings amino acids to ribosome

Genetic Code• Universal across all domains

of life• three bases = codon

• There are 64 possible codons (for 20 possible amino acids).

• The code has "start" and "stop“ codons

• The code was cracked largely by Marshall Nirenberg

• Put synthetic RNA into "cell free" E. coli extract and analyzed the polypeptides that were made.

• Nobel Prize: 1968

1. Initiation

• The mRNA attaches to the ribosome

• Methionine is brought to the start codon (AUG) by the methionine tRNA

• tRNA binding at the ribosome – anti-codon matches with the codon

4. The next codon is now available in the A-site for the next incoming charged tRNA

1. The next codon determines the amino acid to be brought

The incoming tRNA enters at the A-site.

2. The growing polypeptide is transfered to the new tRNA molecule. A peptide bond is formed.

3. The ribosome shifts ("Translocates"). The tRNA with the polypeptide is now in the P-site.

The uncharged amino acid is now in the E-site.

3. Termination• When a stop codon (UAG, UAA, or UGA) is

encountered, a release factor binds to the A-site.• The polypeptide chain is released.• The ribosome disassembles.

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

• Since prokaryotes do not have a nucleus, transcription and translation can be coupled.

• Polyribosomes: simultaneous translation of a transcript (even while that transcript is still being made.

Replication

• DNA Helicase unzips DNA• RNA Primers bind to DNA strands• DNA Polymerase adds nucleotides to DNA

– Leading – continuous adding of bases– Lagging – Okazaki fragments– A-T and C-G

• DNA Ligase fills in gaps

Transcription

1. Unzip DNA (helicase)2. RNA Polymerase binds to synthesize RNA3. Match up bases to one strand of DNA

1. Uracil instead of thymine

4. mRNA detaches from the DNA5. mRNA moves out of nucleus and into cytoplasm

Translation

• mRNA attaches to ribosomes• tRNA moves into ribosome• Anticodon matches with mRNA strand and

adds an amino acid– tRNA leaves ribosome

• Stop codon is reached & amino acid chain (polypeptide) detaches from ribosome– Folds and creates a protein

Mutations

• It becomes clear how changes in DNA can affect changes in protein structure, and in physiology. There are 2 major types of DNA-level mutations:

– Point mutations: One DNA base is replaced by another DNA base.

– Frame-shift mutations: DNA bases are inserted or deleted ("in/dels").

• Each type of mutation can have different effects, depending on the situation.

Point Mutations

• Silent - substitution changes a codon to another codon for the same amino acid.

• Missense - substitution changes a codon to a codon for a different amino acid

• Nonsense- substitution changes a codon to a stop codon

Frameshift

Shift the codons

• Insertions – additions of a nucleotide

• Deletion – loss of a nucleotide

• Duplication – repeating sequences of codons