Genetic Code and Protein Synthesis

Week 14

CENTRAL DOGMA

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

E. coli DNA polymerases

Mammalian DNA polymerases

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

DNA replication begins at specific chromosomal sites called replication origins

Consensus sequence of the minimal bacterial replication origin

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
7

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

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 diversity Recombination occurs randomly between two homologous sequences and the frequency of recombination between two sites is proportional to the distance between the site
10

Plasmid DNA in the process of recombination

11

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:
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.
12

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

13

Transcription reaction - RNA polymerase

Nucleophilic attack by 3 hydroxyl group

Requirements: a template, activated precursors (NTPs), & Divalent metal ion, Mg2+ or Mn2+

14

RNA polymerase: instructions from DNA templates

15

mRNA & DNA complementarity

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

16

Prokaryotic promoter
Consensus sequences centered at -10, & -35

Promoter sites specifically binds RNA polymerase, & determine where transcription begins

17

Eukaryotic promoter
Consensus sequences centered at -25 & -75

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
18

Termination signal in E. coli

Sequence at 3 end of mRNA: Hairpin loop followed by a string of uridines (U) Alternatively, transcription ended by action of Rho protein

19

mRNA modification in eukaryotes

(Less known about transcription termination in eukaryotes) mRNA is modified after transcription

A cap structure is attached to 5 end & a sequence of adenylates, the poly(A) tail, is added to the 3 end
20

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.

21

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.

22

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 for same aa, Synonyms differ in last base, 3 stop codons, designate translation termination
23

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.

24

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

25

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

26

27