Day 2

Genetic information, stored in DNA, is conveyed as proteins

In sickle-cell anemia, one nucleotide change is responsible for the one amino acid change.

Sickle-cell anemia is caused by one amino acid change.

A single base-pair mutation is often the cause of a human genetic disease.

Alteration of the primary sequence of the polypeptide may alter the secondary and tertiary sequence of the protein. The altered protein may not function properly.

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Basic amino acid structure:

A protein also has polarity- the N-terminal end and the C- terminal end:

The immediate product of translation is the primary protein structure

The primary sequence dictates the secondary and tertiary structure of the protein

 helical structure is a very regular structure (3.6 amino acids/turn)

 -sheet: anti-parallel

 -sheet: parallel

Two questions Can you change the 3 o (tertiary) sequence without changing the 1 o (primary) sequence? Can you change the 1 o (primary) sequence without changing the 3 o (tertiary) sequence?

List of Amino Acids and Their Abbreviations Nonpolar (hydrophobic) amino acid3 letter code1 letter code glycineGlyG alanineAlaA valineValV leucineLeuL isoleucineIleI methionineMetM phenylalanin ePheF tryptophanTrpW prolineProP

Polar (hydrophilic) serineSerS threonineThrT cysteineCysC tyrosineTyrY asparagineAsnN glutamineGlnQ Electrically Charged (negative and hydrophilic) aspartic acidAspD glutamic acidGluE Electrically Charged (positive and hydrophilic) lysineLysK arginineArgR histidine Others X = unknown HisH * = STOP

The ‘protein-folding problem’. Proteins -- hundreds of thousands of different ones -- are the biochemical molecules that make up cells, organs and organisms. Proteins put themselves together, in a process termed "folding." How they do that is called "the protein- folding problem," and it may be the most important unanswered question in the life sciences.

The transformation happens quickly and spontaneously. It takes only a fraction of a second for a floppy chain of beads to fold into the shape it will keep for the rest of its working life. How does that happen? How do the linear -- and, in some sense, one-dimensional -- structures of proteins carry the information that tells them to take on permanent three- dimensional shapes? Is it possible to study a protein chain and predict the folded shape it will take? That is the protein-folding problem.

DNA sequencing information  predictions of the primary amino acid sequence. Needed- Software that will convert the 1 o sequence to its corresponding 3 o sequence. Needed- Software that will describe a 1 o sequence that will generate a particular 3 o sequence.

WHY IS PROTEIN FOLDING SO DIFFICULT TO UNDERSTAND? It's amazing that not only do proteins self- assemble -- fold -- but they do so amazingly quickly: some as fast as a millionth of a second. While this time is very fast on a person's timescale, it's remarkably long for computers to simulate. In fact there is a 1000 fold gap between the simulation timescales (nanoseconds) and the times at which the fastest proteins fold (microseconds).

A Glimpse of the Holy Grail? The prediction of the native conformation of a protein of known amino acid sequence is one of the great open questions in molecular biology and one of the most demanding challenges in the new field of bioinformatics. Using fast programs and lots of supercomputer time, Duan and Kollman (1) report that they have successfully folded a reasonably sized (36-residue) protein fragment by molecular dynamics simulation into a structure that resembles the native state. At last it seems that the folding of a protein by detailed computer simulation is not as impossible as most workers in the field believe.1

Proteins from Scratch: Not long ago, it seemed inconceivable that proteins could be designed from scratch. Because each protein sequence has an astronomical number of potential conformations, it appeared that only an experimentalist with the evolutionary life span of Mother Nature could design a sequence capable of folding into a single, well-defined three-dimensional structure. But now, on page 82 of this issue, Dahiyat and Mayo (1) describe a new approach that makes de novo protein design as easy as running a computer program. Well almost.821

Progress in the ‘protein-folding problem’? When proteins fold, they don’t try ever possible 3D conformation. Protein folding is an orderly process (i.e. there are molecular shortcuts involved).

Success in protein-folding? Given the primary sequence of a protein, the success rate in predicting the proper 3D structure of a protein shows strong correlation, to the % of the protein that showed similarity to proteins of known structure.

Genomics Research Funding (selected programs; $ millions) PROGRAM NHGRI (U.S.) WELCOME TRUST (U.K.) STA (JAPAN)39115 ENERGY (U.S.) 8589 GHGP1979 SWEDEN535

Link to NCBINCBI

How to find a gene? The simplest way is too search for an open reading frame (ORF). An ORF is a sequence of codons in DNA that starts with a Start codon, ends with a Stop codon, and has no other Stop codons inside.

Finding a gene is much more difficult in eukaryotic genomes than in prokaryotic genomes. WHY??

mid 1970s- The discovery of ‘split genes’. Split genes are the norm in eukaryotic organisms.

Exon = Genetic code Intron = Non-essential DNA ? ?

The mechanism of splicing is not well understood.

Alternate Splice sites generate various proteins isoforms

Splicing mutants do exist.

. Most mutations in introns are (apparently) harmless Consequently, intron sequences diverge much quicker than exons.

Prokaryotic cells- No splicing (i.e. – no split genes) Eukaryotic cells- Intronless genes are rare (avg. # of introns in HG is 3-7, highest # is 234)

How to confirm the identification of a gene? Answer- Identify the gene by identifying its promoter.

Promoters are DNA regions that control when genes are activated.

Exons encode the information that determines what product will be produced. Promoters encode the information that determines when the protein will be produced.

Demonstration of a consensus sequence. De

How to find a gene? Look for a substantial ORF and associated ‘features’.

The End