Genetic Code and Protein

SynthesisSynthesis

Week 14

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CENTRAL DOGMA

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Replication

• The process by which copies of the DNA are made

• Must have very high fidelity or accuracy to ensure proper

function of gene products and healthy offspring

• DNA replication is accomplished by DNA polymerases• DNA replication is accomplished by DNA polymerases

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E. coli DNA polymerases

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Mammalian DNA polymerases

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The DNA region to be replicated is copied by what isreferred to as a semi-conservative mechanism and

bidirectional

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DNA replication begins at specific chromosomal sites called replication origins

Consensus sequence of the minimal bacterial replication origin

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Replication origins, regardless of organism, are (1) unique DNA segments with multiple short repeats, (2) recognized by multimeric origin-binding proteins, and (3) usually contain an A-T rich stretch

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DNA Repair

• Errors in DNA replication can produce a variety of mutations by failure of proofreading proofreading mechanisms

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Recombination between homologous DNA sites

• Recombination provides a means by which a genome can change to generate new combinations of genes

• Homologous recombination allows for the exchange of blocks of genes between homologous chromosomes and thereby is a mechanism for generating genetic and thereby is a mechanism for generating genetic diversity

• Recombination occurs randomly between two homologous sequences and the frequency of recombination between two sites is proportional to the distance between the site

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Plasmid DNA in the process of recombination

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The Transcription

• Transcription is catalyzed by DNA-dependent RNA polymerases, which use ribonucleoside 5-triphosphates to synthesize RNA complementary to the template strand of duplex DNA

• Three major kinds of RNA are produced: • Three major kinds of RNA are produced:

– Messenger RNAs (mRNAs) encode the amino acid sequence of one or more polypeptides specified by a gene or set of genes.

– Transfer RNAs (tRNAs) read the information encoded in the mRNA and transfer the appropriate amino acid to a growing polypeptide chain during protein synthesis.

– Ribosomal RNAs (rRNAs) are constituents of ribosomes, the intricate cellular machines that synthesize proteins.

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Transcription occurs in several phases: binding of RNA polymerase to aDNA site called a promoter, initiation of transcript synthesis, elongation,and termination.

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Transcription reaction - RNA polymerase

Nucleophilic attack by 3’ hydroxyl group

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Requirements: a template, activated precursors (NTPs), &

Divalent metal ion, Mg2+ or Mn2+

RNA polymerase: instructions from DNA templates

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mRNA & DNA complementarity

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mRNA sequence is the compliment of that of the DNA template

& is the same as that of the coding DNA strand, except for T in

place of U

Prokaryotic promoter

Consensus sequences centered at -10, & -35

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Promoter sites specifically binds RNA polymerase,

& determine where transcription begins

Eukaryotic promoter

Consensus sequences centered at -25 & -75

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Eukaryotic promoters are further stimulated by

enhancer sequences (can be at a distance of several kb from

start site on either its 5’ or 3’ side

Termination signal in E. coli

Sequence at 3’ end

of mRNA:

Hairpin loop

followed by a

string of uridines (U)

Alternatively,

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Alternatively,

transcription ended

by action of

Rho protein

mRNA modification in eukaryotes

(Less known about transcription termination in eukaryotes)

mRNA is modified after transcription

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A “cap” structure is attached to 5’ end & a sequence of

adenylates, the poly(A) tail, is added to the 3’ end

The Genetic Code

• The particular amino acid sequence of a protein is constructed through the translation of information encoded in mRNA. This process is carried out by ribosomes.

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Amino acids are specified by mRNA codons consisting of nucleotide triplets. Translation requires adaptor molecules, the tRNAs, that recognize codons and insert amino acids into their appropriate sequential positions in the polypeptide.

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Genetic code, degenerate (64 codons, 20 aas)

Trp & Met, one codon

each,

other 18 aas, two or

more codons,

Leu, Arg, & Ser, six

codons each,

Synonyms, codons

2323

Synonyms, codons

for same aa,

Synonyms differ in

last base,

3 stop codons,

designate translation

termination

The Translations

• Ribosomes are the RNA-protein complexes that catalyze the polymerization of amino acids bound to aminoacyltRNA molecules. All ribosomes are composed of two subunits; prokaryotic ribosomes contain three rRNA molecules, and eukaryotic ribosomes contain four.

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• The growing polypeptide chain is attached to a tRNA in the peptidyl (P) site of the ribosome, and the aminoacyl-tRNA molecule bearing the next amino acid to be added to the nascent polypeptide chain docks in the aminoacyl (A) site.

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Translation begins with the formation of an initiation complex consisting of an initiator tRNA, the mRNA template, the ribosomal subunits, and several initiation factors.

• In prokaryotes, initiation occurs just downstream of Shine-Dalgarno sequences; in eukaryotes, initiation usually occurs at the initiation codon closest to the end of the mRNA

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