City College of San Francisco
Microbiology 12
Topics to be covered in this lecture include:
Genetics is the study of informational macromolecules such as DNA and RNA and the unit of heredity known as the gene. Genes are sequences of nucleotides within DNA that code for functional proteins. The field of genetics also analyzes the arrangement of genes within organisms (genotype) and how these genes are expressed (phenotype).
Although the majority of organisms carry DNA as their genetic material there are some important exceptions to this rule in the microbial world:
The flow of genetic information
Prior to the development of the microscope how organisms carried out inheritence remained a mystery. Preformist theory advocated that minature "preformed" copies of human beings known as "homunculi" were carried within sperm and that the egg was just a source of nutrients for the little man to be! This theory is not as crazy as it may first seem. The sperm nucleus does carry a partial "blueprint" of a being to be...but that being is not preformed; the blueprint is the genetic instructions needed to form the organism.
In order for stable inheritance to occur there must be an organized flow of information, in the form of genes, from one generation to the next. The central dogma of molecular biology holds that when a gene is expressed the information in:
DNA is TRANSCRIBED(copied) into a messenger RNA form. In turn the information stored in mRNA is TRANSLATED into a functional protein
Well, I have always been a bit of a rebel and generally believe that if a law exists it will eventually be broken by someone or something! Maybe that's why I love microbes. Microbes often break "our rules."
Key figures and achievements in the field of genetics: (This list is far from complete!)
Gregor Mendel: the father of classical genetics. In 1865, the Czech monk reported his discoveries on the mechanism of heredity to the Brunn Society of Natural Science. Mendel's work elegantly complemented the theories of evolution and natural selection proposed by Charles Darwin. Mendel's work was largely ignored until the turn of the century. For over 8 years Mendel carried out hybridization experiments using the garden pea. Mendel also followed the scientific method in a logical manner keeping accurate records of his work and foccusing on one inherited trait at at time in an experiment.
Frederick Griffiths:demonstrated in the late 1920's that killed virulent pneumococci could "transform" avirulent pneumococci into pathogenic bacteria.
Oswald Avery, Colin MacLeod and Maclyn McCarty:confirmed Griffith's experiments. In 1944 they published a landmark paper reporting that the "transforming principle' was DNA. The researchers never received a Nobel prize for their breakthrough observation.
With the exception of Rosalind Franklin, all of the following scientists were awarded the Nobel Prize for research in microbial genetics:
George Beadle and Edward Tatum, Joshua Lederberg: Published a scientific paper in 1940 describing the "One gene-one enzyme hypothesis." These investigators answered a critical question of how genes function by studying a common bread mold known as Neurospora crassa. By studying mutants of this mold, the reseachers were able to establish that a different enzyme was used at eash step in a biochemial pathway involving the breakdown of glucose.
James Watson, Francis Crick and Maurice Wilkins: Received a Noble prize in 1962 for their infamous research on the structure of DNA which was published in 1953. Many believe that Rosalind Franklin deserved to share the prize as her X-ray crystallography studies of DNA provided many important clues about its structure.
Jacques Monod and Francois Monod: Awarded Nobel prize in 1965 for describing how protein syntheis is regulated in bacteria using the "lac operon" model.
Renatto Dulbecco, Howard Temin and David Baltimore:Awarded Nobel Prize in 1975 for discovery of reverse transcriptase and description of cancer-causing RNA viruses
Paul Berg: Received Nobel Prize in 1980 for experiments in genetic engineering and gene splicing.
Barbara McClintock:Received Nobel Prize in 1983 (at the age of 81!) for her discovery of transposons ("jumping genes") in corn. Transposons are small DNA elements that can move between chromosomes and plasmids.
J.M. Bishop and Harold Varmus: Awarded the Nobel Prize in 1989 for their discovery of cancer-causing oncogenes
Kary Mullis: Awarded the Nobel Prize in 1993 for his development of the Polymerase Chain Reaction (PCR) used to amplify DNA.
For a review of nucleic acid structure and images click on this biological macromolecules lecture
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) share many features in common but they do exhbit some important differences:
Describes the relationship between the nucleotide-base sequence of DNA, the corresponding codons (units of genetic information) in mRNA, and the amino acids for which the codons code.
The genetic code or language is universal. Viruses and all living organisms expoit the same four-letter alphabet to spell out their genetic instructions. Superficially, we could not tell based on the A, T, C, G alphabet if we were dealing with a virus, a fruit-fly or a human being. What is important is how the alphabet and genes are arranged. It is how the letters of the genetic alphabet are arranged that spells out the nature of an organism.
The instructions that are necessary to build a protein are contained in messenger RNA in the form of "triplet codons." A particular arrangement of three bases in mRNA dictates a specific amino acid. There are 64 codons in total.
The code is described as redundant or degenerate: Some amino acids are coded for by more than one codon. For example, the codons UUU and UUC both code for the SAME amino acid, phenylalanine.
Not all codons code for amino acids (the 61 codons that do are called sense codons). Three codons that do not code for amino acids are termed stop or nonsense codons. They are UAG, UGA, and UAA.
Just as we use punctuation to help us read our own language, so too does the language of the genetic code. We need to be told where to start reading a sentence and when to stop. We could not do this without punctuation.
The genetic code is analagous to a series of three letter words:
The fat cat can eat.
We know the start of the sentence is signified by a capital letter. Similarly, the Start codon AUG always codes for met (methionine) and signifies start of protein synthesis.
We know that the end of a sentence is indicated by a period. Stop codons serve this purpose for the genetic code and signify the end of protein synthesis.
Prokaryotic (bacterial) DNA differs from eukaryotic DNA in some fundamentally important ways:
Bacterial DNA is usually arranged a circular, double stranded molecule which is tightly coiled (supercoiled) and attached to the inner face of the plasma membrane.
In prokaryotes , the coiling of DNA circles is introduced by an enzyme known as DNA gyrase (this enzyme is the target of quinolone antibiotics which inhibit this enzyme in bacteria). In eukaryotes, the coiling of DNA is the result of chromatin structure; DNA is wound into a complex with histone proteins.
Bacterial DNA lies within the cytoplasm in a nucleoid region but is not contained within a nucleus or delimited by a nuclear envelope.
Each DNA strand acts as a template which can be copied into a complementary strand of either DNA (replication) OR mRNA (transcription). As an analogy think of how you develop your photographs. The information in your negatives allows you to reproduce a positive print of an image. In each case the information is complementary or equivalent.
Each strand of DNA is aligned in an 'antiparallel' fashion. That is, each strand is in an opposite orientation from the other. Strands are bonded to each other by the rules of complementary base-pairing. The orientation and bonding of the two DNA strands is analogous to two passenger trains pasing each other on opposite railroad tracks. The hydrogen bonds are somewhat like the passengers on each train shaking hands.
Before DNA can be replicated the DNA strands must be separated and hydrogen bonds (chemical 'handshakes') must be broken. Enzymes called helicases unwind the DNA.
In bacteria, the double helix separates at a specific region called the origin of replication. This site marks the start of synthesis of new DNA. To prevent the separated DNA strands from annealing or joining back together prematurely single-stranded binding proteins (SSB proteins) bind to the separated DNA strands. In bacteria two replication forks move in opposite directions around the circular chromosome (bidirectional replication).
one new strand and for this reason the replication process is called semiconservative. This mechanism was figured out by the experiments of Matthew Meselson and Franklin Stahl. A bacterial culture was grown over many generations in a medium containing radiolabeled 15N. Cells were then transferred to a medium containing the common isotope of nitrogen, 14N. After several generations of replication, cells were removed and their DNA strands isolated. They demonstrated that the semiconservative mode of replication was correct.
The major polymerase in E.coli that is responsible for the advance of the replication fork is DNA polymerase III. This enzyme builds DNA using the four deoxynucleoside 5' triphosphates available in the cytoplasmic pool. (Another enzyme known as DNA polymerase I acts in a similar manner but has less of a role to play in E.coli). DNA polymerase III has a complex structure and the holoenzyme consists of numerous subunits.
The following requirements have to be met in order for DNA polymerase to function:
DNA polymerase I has several other important activites:
If both daughter strands grew in the same direction, only one strand has a free 3'-OH group as the two strands are antiparallel. The other strand would have a free 5' phosphate group and this can not be used by polymerases. Consequently, DNA synthesis on the lagging strand ocurs in fragments which are then spliced together.
A summary of the events on the lagging strand follows:
The whole process is rather like editing a film-strip. The pieces of film that are not wanted are clipped out and the part of the film that makes sense is joined together.
Transcription is the process whereby RNA molecules are initiated, elongated and terminated using a DNA template.
Initiation of transcription
To start transcription, RNA polymerase binds to particular sequences in DNA called promoters. These sequences are known as consensus sequences and many E. coli promoters possess variations of a common sequence (TATAAT). RNA synthesis proceeds a short distance downstream from this recognition site.
There are both strong promoters and weak promoters. Those that are strong promoters produce more copies of mRNA and thus more copies of protein per cell because recognition and/or binding by RNA polymerase is greater.
Chain elongation
A summary of the key features of prokaryotic RNA synthesis:
Chain termination
mRNA synthesis is terminated at specific base sequences within the DNA molecule and RNA polymerase dissociates from the DNA. These sequences are of two types:
Translation is the process whereby the information stored in mRNA is 'decoded' in order to build a functional protein. Thus, the base sequence of a DNA molecule determines the amino acid sequence of a protein.
A DNA sequence that corresponds to one polypeptide is called a cistron. In prokaryotes it is common for mRNA to be polycistronic; that is, it codes for several different polypeptides. A typical polycistronic mRNA is between 3,000-8,000 nucleotides. This is a way for bacteria to control production of related proteins that often participate together in a single metabolic pathway.
The mRNA that corresponds to a DNA cistron is known as a reading frame as it is 'read' by the protein-producing machinery of the cell.
The basic ingredients for bacterial protein synthesis are:
Like transcription, translation also has three main stages: initiation, chain elongation and termination.
Initiation
Chain elongation
Termination
When a protein is completed it is released from the mRNA/ribosomal complex. This is achieved by the presence of punctuation marks or 'stop codons' in the mRNA which signal termination of protein synthesis. Instead of one period marking the end of a sentence, mRNA has three possible stop codons: UAA, UAG and UGA.