Chapter 9
Microbiology Review Sheet

WHAT IS GENETICS

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:

  • Certain virus families possess RNA versus DNA as their genetic material. The influenza viruses and HIV, the virus that causes AIDS, are important examples of RNA viruses.
  • Infectious agents known as PRIONS are believed to consist entirely of protein and to date, are not known to be associated with any form of nucleic acids

THE FLOW OF GENETIC INFORMATION

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."

  • Take for example the retroviruses. Not only do these viruses possess RNA instead of DNA as their genetic material but they have the ability to copy their genetic material BACKWARDS (retro) into a DNA copy!
  • As already stated, PRIONS seem to break all the conventions of inheritance.

HISTORICAL BACKGROUND

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 synthesis is regulated in bacteria using the "lac operon" model.

NUCLEIC ACID STRUCTURE

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) share many features in common but they do exhibit some important differences:

  • DNA typically occurs as a double-stranded molecule whereas RNA is typically single-stranded (with the exception of some viruses...see I told you that rules are usually broken!)
  • DNA contains the nitrogenous base thymine; RNA contains uracil
  • DNA contains the pentose sugar deoxyribose; RNA contains ribose
  • RNA is often not as long as DNA
  • There are three major forms of RNA (discussed under the section on translation)

THE GENETIC CODE

  • 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 analogous to a series of three letter words.
  • 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.

DNA REPLICATION

  • 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 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).
  • 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:

  • A template strand of DNA must be available to be copied
  • A primer must be available. A primer is a short stretch of nucleotides hydrogen-bonded to the template and whose terminal 3'-OH group is "free."
  • The 3'-OH group at the end of the growing strand participates in a reaction with an incoming dNTP forming a phosphodiester bond. When the new nucleotide is added, its 3"OH is free to participate in the next reaction.
  • All known polymerases (DNA and RNA) are only capable of synthesizing nucleic acids in the 5" ______3" direction. DNA is synthesized in one direction (5' to 3') SENSE STRAND. At the replication fork , one daughter strand (leading strand) is made continously while the other is made discontinously (lagging strand).

DNA polymerase I has several other important activites:

  • It has the ability to proofread newly synthesized DNA and edit out mistakes. It can move in the 3'______5' direction to cut out mismatched bases and it can replace the correct base(s). This is known as a 3'______5" exonuclease activity.
  • Pol I also has 5'______3' exonuclease activity. The main function of this activity is to remove RNA primers from the lagging strand of DNA.

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.

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

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:

  • Four nucleoside 5'-tripohosphates are ATP, GTP, CTP, and UTP (contains uracil). The ribose sugar contains two free -OH groups.
  • A 3'-OH group forms a phosphodiester bond with the 5' triphosphate of an incoming nucleotide. The chain only grows in the 5'______3' direction (this is the same reaction that occurs when building DNA)
  • This process is carried out by RNA polymerase which uses a single strand of DNA as a template.. RNA polymerase does NOT need a primer to initiate chain growth.
  • RNA polymerase in E. coli consists of several subunits
  • The base sequence in RNA is said to be complementary to that contained in DNA. U always pairs with A and G always pairs with C.
  • The nucleotide sequence of the mRNA is read in groups of three bases known as triplet codons.

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:

  • Simple (Rho-independent) sequences that depend only on the DNA sequence
  • Rho-dependent sequences are those that require an accessory termination signal in the form of a protein called Rho which binds tightly to DNA.
  • Note that termination of mRNA synthesis is due to sequences in DNA and is not the same as termination of protein synthesis which is due to stop codons within mRNA.

TRANSLATION

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:

  • messenger RNA: an informational molecule
  • 70S ribosomes (30S and 50S subunits in bacteria)
  • Bacterial ribosomes contain three kinds of rRNA: 5S rRNA, 16S rRNA, and 23S rRNA. As we will see later on, rRNA sequencing is proving very useful to study the evolutionary relationships between bacterial species. It also makes us have to re-evaluate a lot of information!
  • transfer RNA: an informational and structural molecule
  • amino acids

Like transcription, translation also has three main stages: initiation, chain elongation and termination.

Initiation

  • In bacteria protein synthesis begins by the binding of one 30S ribosomal subunit to a mRNA molecule, followed by the binding of the 50S subunit to the 30S subunit to form a 70S initiation complex.
  • Two tRNA binding sites occur in the ribosomal complex. The peptidyl or P site fixes the position at which an incoming tRNA will bind. The aminoacyl or A site is adjacent to the P site and receives the tRNAs to which the growing polypeptide chain is attached.
  • Transfer RNAs are like taxi cabs dropping off passengers at an airport. Once the taxi drops off the customer it is free to pick up more customers and drop them off at the terminus.
  • The initiator tRNA always picks up the amino acid methionine. This tRNA has an anticodon which pairs with the start codon AUG in the mRNA. the tRNA carrying f-methionine positions itself at the P site in the ribosomal complex.

Chain elongation

  • f-Methionine becomes the first protein in the polypeptide chain (although it can be cleaved after translation).
  • Translation of mRNA always occurs in the 5'______3' direction.
  • An incoming tRNA carrying the next amino acid deposits itself in the adjacent A site. Once this occurs an enzyme complex called peptidyl transferase catalyzes the formation of a peptide bond between the amino acid in the P site and that in the A site. .
  • The tRNA in the P site is then free to dissociate from the ribosome and goes back into the cytoplasmic pool to pick up another customer.
  • The mRNA then moves a distance of three bases to position the next codon at the A site
  • These steps are repeated until the final product is assembled

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.