CH 105 - Chemistry and Society

Central Dogma of Biology

08/14/2014

French translation by Kate Bondareva

As mentioned above, each amino acid is specified by a particular combination of three nucleotides in RNA.  The three bases are called a codon.  The Genetic Code consists of a chart which shows what triplet RNA sequence or codon in mRNA codes for which of the 20 amino acids.  One of the codon codes for no amino acids and serves to stop the synthesis of the protein from the mRNA sequence. The genetic code is shown below:

GENETIC CODE

Determining the protein sequence from a DNA sequence.

For a given gene, only one strand of the DNA serves as the template for transcription.  An example is shown below.   The bottom (blue) strand in this example is the template strand, which is also called the minus (-) strand, or the sense strand. It is this strand that serves as a template for the mRNA synthesis. The enzyme RNA polymerase synthesizes an mRNA in the 5' to 3' direction complementary to this template strand. The opposite DNA strand (red) is called the coding strand, the nontemplate strand, the plus (+) strand, or the antisense strand. 

The easiest way to find the corresponding mRNA sequence (shown in green below) is to read the coding, nontemplate, plus (+), or antisense strand directly in the 5' to 3' direction substituting U for T.  Find the triplet in the coding strand, change any T's to U's, and read from the Genetic Code the corresponding amino acid that would be incorporated into the growing protein.  (The strands below are separated into triplets for ease of visualization.)  An example is show below.

DNA 5'  A G G  C C T  T C G  A A C  G G G  A T G  G A A  T G A  3'
DNA 3'  T C C  G G A  A G C  T T G  C C C  T A C  C T T  A C T  5'

RNA 5'  A G G  C C U  U C G  A A C  G G G  A U G  G A A  U G A 3'

PROT N   ARG     PRO    SER    ASP    GLY    MET    GLU     STOP

• DNA or RNA sequence to Amino Acid converter
• reverse translate a protein into a DNA sequence  |  Another reverse translator

END BASIS REVIEW CENTRAL DOGMA OF BIOLOGY

Proteins

In contrast to the linear polymers of DNA and RNA, proteins (linear polymers of amino acids) fold in 3D space to form structures of unique shapes.  Each unique protein sequence (of a given length and sequence of amino acids) folds to a unique 3D shape.  Hence there are about 30,000 proteins of different shapes in humans.   Not only do proteins have unique shapes, but they also have unique nooks and crannies and pockets which allow them to bind other molecules.  Binding of other molecules to proteins or DNA initiates or terminates the function of the protein or nucleic, much like an on/off switch.  The example below show different protein structures, some of which have small molecules or large molecules (like DNA) bound to them.  Some common motifs are found within the 3D structure of the protein.  The include alpha helices and beta sheets.  These are held together by H-bonds between the slightly positive H on the N in the protein backbone and a slightly negative O further away on the protein backbone.   In the Chime models below, use the mouse controls to rotate the molecule.  (Also shift L-mouse click will change the size of the molecule).  Click on the command in the right hand frame to change the rendering of the proteins.  The cartoon view allows a simple way to interpret the overall structure of the main chain.

Mutations

If the DNA sequence in a coding region becomes changed, the resulting mRNA will also be changed, which will lead to changes in the protein sequence.   These changes might have no effect and be silent, if the change in the protein does not affect the folding of the protein or its binding to another important molecule.   However, if the changes affects either the folding or the binding region, the protein may not be able to perform its usual function.   Mutations which substitute nonpolar amino acids for polar/charged ones (or the reverse) have the greatest chance of causing significant changes in structure and/or activity.

• Changes in protein structure/activity depend on place and nature of amino acid change

 If the function was to put on break on cell division, the result might lead the cell to become a cancerous.   Likewise, if the normal protein had a role in causing the cell to die after its intended life span, the cell with the mutant protein might not die and more likely become a tumor.  The opposite scenarios could happen leading the cell to a premature death.  

• Point mutations:  From bad luck and nucleotide analogs
• Point mutations:  From chemical mutagens
• Large mutations:  Deletions, insertions, duplications, and inversions

Check out the model of sickle cell hemoglobin below, which shows how a single base pair change in the DNA can change one amino acid in a protein with lethal consequences.

Jmol Sickle Cell Hemoglobin

Genes and Disease

• A Map showing gene involvement in specific human diseases.  Click on the chromosome number. 

Activation and Repression of Gene Transcription:

Imagine you have been given a one foot string which represents part of a human chromosome.  There just happens to be a small dot in the dead center of the string you have been given. It represents the gene for a particular protein called metallothionein. This gene is expressed and the protein metallothionein is made when cells are exposed to toxic heavy metals like mercury.  It would be as waste of metabolic energy if all 30,000 genes were expressed (transcribed and translated into proteins) all the time.   In the case of dealing with mercury exposure, it would make most sense if the gene for metallothionein, in the absence of mercury,  was not expressed or  expressed to a small, constituitive level, but in the presence of mercury, its level of expression would increase to deal with the threat to the cell.  How might the cell accomplish these two feats:

1. How might gene expression be repressed in the absence of mercury
2. How might gene expression be activated in the presence of mercury

One of the central questions of modern biology is what controls gene expression. As we have previously described, genes must be "turned on" at the right time, in the right cell. To a first approximation, all the cells in an organism contains the same DNA (with the exception of germ cells and immune cells). Cell type is determined by what genes are expressed at a given time. Likewise, cell can change (differentiate) into different types of cells by altering the expression of genes. The central dogma of biology describes how genes are first transcribed to RNA, and then the mRNA is translated into a corresponding protein sequence. Proteins can then be post-translationally modified, localized to certain locales within the cells, and ultimately  degraded. If functional proteins are considered the end-product of gene expression, the control of gene expression could theoretically occur at any of these steps in the process.

•  PROCESSES THAT AFFECT THE STEADY STATE CONCENTRATION OF A PROTEIN 

Mostly, however, gene expression is controlled at the level of transcription. This makes great biological sense, since it would be less energetically wasteful to induce or inhibit the ultimate expression of a functional protein at a step early in the process. How can gene expression be regulated at the transcriptional level? Many examples have been documented. The main control is typically exerted at the level of RNA polymerase binding just upstream (5') of a a site for transcriptional initiation. Other factors, called transcription factors (which are usually proteins), bind to the same region and promote the binding of RNA polymerase at its binding site, called the promoter. Proteins can also bind to sites on DNA (operator in prokaryotes) and inhibit the assembly of the transcription complex and hence transcription. Regulation of gene transcription then becomes a matter of binding the appropriate transcription factors and RNA polymerase to the appropriate region at the start site for gene transcription. Regulation of gene expression by proteins can be either positive or negative.  Regulation in prokaryotes is usually negative while it is positive in eukaryotes.

• Positive and Negative Regulation of Gene Transcription

Most eukaryotic genes have about 5 regulatory sites for binding transcription factors and RNA polymerase. Examples of these transcription factors are show in the figure below.

• Control of globin gene transcription
• Example of transcription complexes
• p53 information from the HHMI
• animation: p53 and gene transcription from the HHMI (loads slowly)

STRUCTURAL FEATURES OF SPECIFIC DNA BINDING SITES

Since RNA polymerase must interact at the promoter site of all genes, you might expect that all genes would have a similar nucleotide sequence in the promoter region. This is found to be true for both prokaryotic (such as bacteria) and eukaryotic genes. You would expect, however, that all transcription factors would not have identical DNA binding sequences. The sequences of DNA just upstream of the start site of the gene that binds protein (RNA polymerase, transcription factors, etc) are called promoters.  The table below shows the common DNA sequence motif called the Pribnow or TATA box found at around -10 base pairs upstream from the start site, and another at -35.  Proteins bind to these sites and facilitate binding of  RNA polymerase, leading to gene transcripton.

Jmol: TATA Box Binding Protein 

Prokaryotic  Promoter Sequences

In addition, in eukaryotes promoters, sequences further upstream called response elements bind specific proteins (such as CREB or cyclic AMP response element binding protein) to further control gene transcription.

Eukaryotic Response Elements  (RE)s

Proteins can interact specifically with DNA through electrostatic, H-bond, and hydrophobic interactions.  AT and GC base pairs have available H bond donors and acceptors which are exposed in the major and minor grove of the ds DNA helix, allowing specific protein-DNA interactions.

Jmol: Simple DNA Tutorial (see last selection buttons to see H bond donors and acceptors in the major grove.

STRUCTURAL FEATURES OF DNA-BINDING PROTEINS

Not any protein can bind specifically to DNA. Analysis of DNA binding proteins shows common motifs are found among them.

• helix-turn-helix: found in prokaryotic DNA binding proteins. The figures shows two such proteins, the cro repressor form bacteriophage 434 and the lambda repressor from the bacteriophage lambda. (Bacteriophages are viruses that infect bacteia.)  Notice how specificity is achieved, in part, by the formation of specific H-bonds between the protein and the major grove of the operator DNA.
• Lambda Repressor/DNA Complex
• H Bond interactions between l repressor and DNA

Jmol Lambda-Repressor Complex

• zinc finger: (eukaryotes) These proteins have a common sequence motif of
  X₃-Cys-X_2-4-Cys-X₁₂-His-X_3-4-His-X_4- in which X is any amino acid. Zn²⁺ is tetrahedrally coordinated with the Cys and His side chains, which are on one of two antiparallel beta strands, and an alpha helix, respectively. The zinc finger, stabilized by the zinc, binds to the major groove of DNA.

Jmol  Zif268:DNA Complex
 

• steroid hormone receptors: (eukaryotes) In contrast to most hormones, which bind to cell surface receptors, steroid hormones (derivatives of cholesterol) pass through the cell membrane and bind to cytoplasmic receptors through a hormone binding domain. This changes the shape of the receptor which then binds to a specific site on the DNA (hormone response element) though a DNA binding domain. In a structure analogous to the zinc finger, Zn ²⁺ is tetrahedrally coordinated to 4 Cys, in a globular-like structure which binds as a dimer to two identical, but reversed sequences of DNA (palindrome) within the major grove. (An example of a palindrome: Able was I ere I saw Elba.)
• leucine zippers: (eukaryotes) These proteins contain stretches of 35 amino acids in which Leu is found repeatly at intervals of 7 amino acids. These regions of the protein form amphiphilic helices, with Leu on one face. Two of these proteins can form a dimer, stabilized by the binding of these amphiphilic helices to one another, forming a coiled-coil, much as in the muscle protein myosin. Hence the leucine zipper represents the protein binding domain of the protein. The DNA binding domain is found in the first 30 N-terminal amino acids, which are basic and form an alpha helix when the protein binds to DNA. The leucine zipper then functions to bring two DNA binding proteins together, allowing the N-terminal bases helices to interact with the major grove of DNA in a base-specific fashion.

Jmol  Leucine Zipper

PHOSPHORYLATION AND CONTROL OF GENE EXPRESSION

A common way to control gene expression is by controlling the phosphorylation of transcription factors by ATP.  This modification might activate or inhibit the transcription factor in turning on gene expression.  The added phosphate groups might be necessary for direct binding interactions leading to gene transcription or they might lead to a conformational change in the transcription factor, which could activate or inhibit gene transcription.  A recent example of this later case is the control of the activity of the transcription factor p53.  p53 has many activities in the cell, a primary one as a suppressor of tumor cell growth.  If a cell is subjected to stress that results in genetic damage (an evident which could lead a cell to transform into a tumor cell), this protein becomes an active transcription factor, leading to the expression of many genes, including those involved in programmed cell death and cell cycle regulation.  Both of these effects could clearly inhibit cell proliferation.  Hence p53 is a tumor suppressor gene.  p53 is usually bound to the protein HDM2 which down regulates its activity by leading to its degradation.  Stress signals lead to the activation of protein kinases in the cell (such as p38, JNK, and cdc2), causing phosphorylation of serine and threnine amino acids in p53 (Ser 33 and 315 and Thr 81).  This leads to the binding of the proteni Pin 1, which changes the shape of p53 and leads to its activation as a transcription factor.

• Activation of p53 as a transcription factor by phosphorylation and conformational change