2.7 DNA replication, transcription and translation Flashcards
Analysis of Meselson and Stahl’s results
- Grew E. coli bacteria in medium containing N15, a heavy isotope of N14
- After 15 generations, they transferred the bacteria to a medium containing only N14
- The bacteria replicated every 20 min, then extracted
- spun it in a centrifuge, DNA created a layer based on its density
results of Meselson and Stahl’s experiment
Their results showed that N14 was incorporated into the new DNA molecules with each new replication.
semi-conservative replication
- Two strands of the double helix separate
- Each original strand acts as a guide or template for the new strand
- New strands are created by adding nucleotides following complementary base-pairing
- The two new strands formed will be identical to the original strand.
helicase
- Unwinds and unzips the DNA Helix
- Separates the two polynucleotide strands by breaking the hydrogen bonds between complementary base pairs
- ATP is needed by helicase to both move along the DNA molecule and to break the hydrogen bonds
- The two separated strands become parent/template strands for the replication process
DNA polymerase
- creates complementary strands from template strands
- DNA polymerase brings nucleotides into position where they can form H-bonds with the template strand
- DNA polymerase always builds in a 5’ to 3’ direction
- DNA polymerase bonds the 5’ (phosphate) end of the free nucleotide to the 3’ (hydroxyl) end of the new strand in a condensation reaction
- DNA polymerase catalyses the covalent phosphodiester bonds between sugars and phosphate groups
- DNA Polymerase proof-reads the complementary base pairing.
Energy is needed to…
link new nucleotides to the growing DNA strand
deoxynucleoside triphosphates…
- align opposite their matching base partner
- DNA plolymerase cleaves the 2 excess phosphates, energy released is used to link phosphate to 3’ end of sugar covalently
Polymerase Chain Reaction (PCR)
- Denaturation: DNA sample is heated to separate it into two strands
- Annealing: DNA primers attach to opposite ends of the target sequence
- Elongation: A heat-tolerant DNA polymerase (Taq) copies the strands
Transcription
the synthesis of RNA, using DNA as a template
-happens in the nucleus of eukaryotic cells
- happens in the cytoplasm of prokaryotic cells
Translation
when a ribosome reads the mRNA and builds the corresponding polypeptide chain
- happens in the cytoplasm for both eukaryotic and prokaryotic cells
Three main types of RNA
Messenger RNA (mRNA): A transcript copy of a gene used to encode a polypeptide
Transfer RNA (tRNA): A clover leaf shaped sequence that carries an amino acid
Ribosomal RNA (rRNA): A primary component of ribosomes
transcription process
- enzyme RNA polymerase binds to site on DNA at start of a gene
- RNA polymerase separates the DNA strands and synthesises a complementary RNA copy from the antisense DNA strand
- RNA polymerase forms cvalent bonds between RNA nucleotides
- Eventually, RNA polymerase comes across a termination sequence in the DNA, RNA polymerase will detach, double helix reforms
Introns
do not code for proteins and must be removed before the mRNA is considered mature
exons
(the coding part) are spliced back together, the completed mRNA strand leaves the nucleus.
A ribosome is composed of…
two halves, a large and a small subunit. During translation, ribosomal subunits assemble together like a sandwich on the strand of mRNA:
Only certain genes in a genome need to be expressed depending on:
Cell specialism
Environment
The genetic code
the set of rules by which information encoded in mRNA sequences is converted into proteins (amino acid (AA) sequences) by living cells
Codons are a triplet of bases which…
encodes a particular AA
- The codons can translate for 20 amino acids
The coding region always starts with a START codon
(AUG)
Methionine
The coding region of mRNA terminates with a STOP codon
the STOP codon does not add an amino acid – instead it causes the release of the polypeptide
Amino acids are carried by
transfer RNA (tRNA)
The anti-codons on tRNA are complementary to the codons on mRNA
tRNA molecules
- have an anticodon of three bases that binds to a complementary codon on mRNA
- carry the amino acid corresponding to their codon
mRNA has a sequence of…
codons that specifies the amino acid sequence of the polypeptide
Ribosomes:
act as the binding site for mRNA and tRNA
catalyse the peptide bonds of the polypeptide
An outline of translation and polypeptide synthesis:
- An mRNA binds to the small subunit of the ribosome
- A molecule of tRNA binds to the ribosome. This tRNA has an anti-codon that is complementary to the first codon on mRNA → That codon is always AUG, which codes for the amino acid “met” (methionine)
- A second tRNA with an anticodon complementary to the second codon binds. Only 2 tRNA molecules can bind to the mRNA at the same time.
- A peptide bond is created between the two amino acids – a dipeptide forms
- The ribosome moves along the mRNA like a roller coaster on a track… it shifts over one codon, and the next tRNA carrying the appropriate amino acid comes in and binds to that codon with its anti-codon.
- This process repeats over and over again until a STOP codon is reached. This doesn’t code for an amino acid, but rather, initiates the detachment of the polypeptide.
- This polypeptide (amino acid chain) can now go on to fold and produce a functional protein
produce human insulin in bacteria
- The gene responsible for insulin production is extracted from a human cell
- It is spliced into a plasmid vector (for autonomous replication and expression) before being inserted into a bacterial cell
- The transgenic bacteria (typically E. coli) are then selected and cultured in a fermentation tank (to increase bacterial numbers)
- The bacteria now produce human insulin, which is harvested, purified and packaged for human use (i.e. by diabetics)
