Download Nucleotide is composed of a ribose sugar, a base and a phosphate

MIC2MI notes
Nucleic acid structure:
• Nucleotide is composed of a ribose sugar, a base and a phosphate
• Nucleotides are joined together by a 5' to 3' phosphodiester bond
• Purines (A, G) and pyrimidines (C, T, U)
• G-C triple hydrogen bonds, A-T double hydrogen bonds
• RNA has an OH group, DNA has an H group (deoxy)
• RNA is single stranded, DNA is double stranded (antiparallel)
Bacterial genome:
• Single, closed circular DNA molecule
• DNA is supercoiled by topoisomerase, in order to fit into the bacterial cell
• Genes are organised into clusters (operons)
• Can contain extra-chromosomal elements
• DNA polymerase III – main DNApol involved in replication (catalysis the incorporates of the
nucleotides into the growing DNA strand)
• DNA polymerase II – DNA repair and a minor role in replication
• DNA polymerase I – DNA repair and SOS responses. Fills gap from removed RNA primer in
lagging strand.
DNA replication:
• Semi-conservative, each new DNA molecule consists of one parental strand and one newly
synthesised strand.
• Synthesised by DNA polymerase III – requires Mg 2+ ions as co-factors for enzyme function.
• Replication is bidirectional from origin of replication
• Unincorporated nucleotides = deoxyribonucleotide triphosphate, incorporated nucleotide =
deoxyribonucleotide monophosphate
DNA strands: The start of the gene is always closest to the 5' end
• Template strand/ non-coding strand/ antisense (3' to 5') strand – The template used for transcription
• Non-template strand/ coding strand/ sense (5' to 3') strand – Codes for the gene. Same sequence as
the new RNA strand (T replaced with U)
RNA molecules:
• mRNA – Encodes the information for the synthesis of proteins (translated)
• rRNA – A structural component of ribosomes (not translated)
• tRNA – Adapter molecule between mRNA and ribosomes, that carries the amino acids to the
ribosome for the translation of mRNA (not translated)
Transcription:
• Mediated by RNA polymerase
• Transcription is initiated at the promotor site, which contain 2 highly conserved sequences
◦ Pribnow box or -10 (TATAAT box) located 10bp upstream of the transcription initiation site
◦ -35 bp sequence (TTGACA) located 35 bp upstream of the transcription initiation site
• Promotor sites are recognised by sigma factors (RNA pol + sigma factors = holoenzyme). Different
sigma factors will recognise different types of promotors ( 70 is the standard sigma factor of most
bacterial genes, 32 for the heat shock gene)
• Sigma factor is released when transcription begins
Translation:
• Decoding of mRNA to make a protein
• Open Reading Frame (ORF) – A continuous stretch of DNA beginning with a start codon, usually
methionine (ATG), and ending with a stop codon (TAA, TAG or TGA in most genomes).
• MRNA is read in triplets called codons
• Third position degeneracy in amino acid codes. In some cases the third nucleotide doesn't have to be
complimentary but will still code for the same animo acid (wobble base)
• Complimentary base pairing between the codon on the mRNA strand and the anticodon on the
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tRNA.
Ribosomes consist of a large (50S) and a small (30S) subunit.
A sequence within the 16S of the small subunit is complimentary to the Shine-Delgarno sequence in
the mRNA (ribosome binding site). Base pairing between the mRNA and rRNA hole the complex
together.
Once the large ribosomal subunit is recruited, the start codon can be recognised and translation is
initiated.
In bacteria, translation can be initiated while mRNA is being synthesised as both are present in the
cytoplasm.
Product
Subunits used
Protein
Synthesis
Site of initiation
Template
Replication
DNA
Transcription
RNA
Translation
Protein
Deoxyribonucleotide
triphosphates
DNA polymerase
Ribonucleotide
triphosphates
RNA polymerase
Amino Acids
5' to 3' direction (cannot
be initiated de novo)
Origin or replication (one
per chromosome)
5' to 3' direction (can be
initiated de novo)
Promotors (several per
chromosome)
Reads RNA in a 5' to 3'
direction
Start codon (binding to
Shine-Delgarno
sequence)
Both strands
Template/ non-coding/
antisense strand
mRNA
Ribosome
Genetic recombination – rearrangment/exchange of genetic material between two sources
suitable media for isolation of strains. In bacteria, exchange of DNA from another cell. Consequences of
recombination include new genotypes and phenotypes, eg. Ability to synthesis a new enzyme, antibiotic
resistance. Strains carrying recombinant DNA are termed recombinants
Homologous recombination – Genetic exchange mediated by identical/almost identical sequences in both
DNA molecules.
• Endonuclease cut/nicks a single DNA strand in each molecule (cleave phosphodiester bonds)
• DNA helicases displaces the nicked DNA strand and single stranded binding proteins attach
• RecA (recombinase) protein mediates base pairing between donor and recipient strands.
• Resolution of DNA molecules by resolvase
• Exonuclease removes mismatches and DNA pol makes repairs
• DNA strands are rejoined by DNA ligase
Recombination with linear DNA:
• Requires two homologous sites
• Donor DNA is integrated between the 2 homologous sequences within the chromosome
• Excised recipient DNA is lost and degraded (does not contain an origin or replication)
• eg. transformation following uptake of naked DNA
Recombination with circular DNA:
• Requires only one recombination event
• Donor DNA is integrated into the chromosome, no loss of chromosomal DNA
• DNA is gained by the recipient
• Circular DNA can be excised as a complete DNA sequence as well
• eg. plasmid integration
Isolation of recombinants - Media must select agains both the donor and the recipient cells, but allow the
growth of the recombinants.
Horizontal Gene Transfer – The exchange of genetic material between bacterial cells. The donor DNA
must be transferred into the recipient before recombination can occur.
• Transformation – Transfer of DNA from the environment
• Transduction – Transfer of DNA from donor to recipient via a bacteriophage