MICROBIAL GENETICS

CHROMOSOME - ONE LONG DOUBLE HELICAL STRAND OF DNA. THE DNA IS TIGHTLY WOUND UP AND ASSOCIATED WITH MANY DNA BINDING PROTEINS. THESE PROTEINS ARE ESSENTIAL FOR CARRYING OUT THE TWO FUNCTIONS OF THE DNA:

(1) REPLICATION

(2) EXPRESSION - WHICH CAN BE DIVIDED INTO TWO PARTS:

(A) TRANSCRIPTION

(B) TRANSLATION

THE DNA BINDING PROTEINS INCLUDE

GENOME - ALL THE GENETIC THAT A CELL MUST HAVE TO FUNCTION AND REPRODUCE AS A CELL OF A PARTICULAR SPECIES.

(E. COLI HAS ONE CIRCULAR CHROMOSOME. THAT ONE CHROMOSOME IS ITS GENOME. THAT ONE CHROMOSOME IS APPROXIMATELY 4,000,000 BASE PAIRS LONG.

THE HUMAN GENOME CONSISTS OF 22 DIFFERENT AUTOSOMAL CHROMOSOMES AND 2 DIFFERENT SEX CHROMOSOMES. MOREOVER, EACH HUMAN CELL (EXCEPT FOR SPERM CELLS AND EGG CELLS) HAS A PAIR OF EACH AUTOSOME AND A PAIR OF SEX CHROMOSOMES (2 X'S OR 1Y PLUS 1X). THEREFORE, HUMAN CELLS ARE SAID TO HAVE A DIPLOID GENOME. BACTERIA HAVE A HAPLOID GENOME.

 

GENE -- A SEQUENCE OF DNA WHICH CODES FOR A FUNCTIONAL PRODUCT. THE PRODUCT IS USUALLY A PROTEIN OF SOME KIND LIKE AN ENZYME OR A RECEPTOR OR A STRUCTURAL PROTEIN.

 

GENOTYPE -- THIS REFERS TO THE GENES WHICH AN ORGANISM ACTUALLY POSSESSES. THIS IS THE "POTENTIAL" OF THE ORGANISM.

 

PHENOTYPE -- THIS REFERS TO THE GENES WHICH ARE ACTUALLY "EXPRESSED" BY THE ORGANISM. THIS IS USUALLY WHAT THE ORGANISM "LOOKS LIKE."

 

ALLELES -- THESE ARE THE DIFFERENT VARIETIES OF A GENE WHICH EXIST IN THE POPULATION OF THE ORGANISM.

 

example: In the human ABO blood group system the gene (the locus) is called I. There are three alleles: IA, IB, and IO. Each individual human can have at most 2 alleles of this gene (because we have only ONE PAIR of the chomosome which carries the I-locus.) Therefore the possible genotypes are:

                                     IA IA                                        
                                     IA IO                                        
                                     IA IB                                        
                                     IB IB                                        
                                     IB IO                                        
                                     IO IO                                        
 
 

With these genotypes, the possible phenotypes would be:

                                       A                                          
                                       B                                          
                                       AB                                         
                                       O                                          
 
 


HOW DNA FUNCTIONS

REMEMBER THAT DNA IS DOUBLE STRANDED.:

THE TWO STRANDS ARE COMPLIMENTARY -- THE STRANDS ARE HYBRIDIZED TO EACH OTHER BECAUSE OF BASE PAIRING.

THE TWO STRANDS ARE ANTI-PARALLEL. (See fig. 8.3 on pg. 193).

FOR EACH GENE, ONE STRAND IS (+) AND THE OTHER IS (-). THE (+) STRAND IS USED TO MAKE THE mRNA.

 

DNA REPLICATION: (See pg. 192, fig 8.2; pg. 194, fig. 8.4; pg. 195, fig. 8.5).

WHAT IS MEANT BY LEADING STRAND AND LAGGING STRAND. WHERE DO RNA PRIMERS FIT INTO THIS?

REPLICATION WILL NEED DNA POLYMERASE, RNA POLYMERASE, HELICASE, GYRASE, LIGASE.

 

THE REPLICATION PROCESS INSURES THAT WHEN DNA IS REPLICATED, TWO EXACT COPIES WILL BE MADE. IF A MISTAKE IS MADE IT IS CALLED A MUTATION. A MUTATION IS AN INHERITABLE CHANGE IN THE NUCLEOTIDE BASE SEQUENCE.

 

MANY BACTERIA PRODUCE RESTRICTION ENDONUCLEASES (RESTRICTION ENZYMES). THESE ENZYMES HAVE BECOME VERY IMPORTANT TOOLS IN GENETIC ENGINEERING. (see pg. 230 in Tortora and pgs. 259, 270 and 304 in Lewis.)

 

THESE ENZYMES OFTEN CUT THE DOUBLE STRANDED DNA AT "PALLINDROME" SEQUENCES IN SUCH A WAY TO GENERATE "STICKY ENDS." SUCH EASILY HYBRIDIZED STRUCTURES ALLOW MOLECULAR GENETICISTS TO CONNECT DNA FRAGMENTS TOGETHER, SPLICING GENES FROM DIFFERENT SOURCES.

DNA EXPRESSION

enhancer

promotor

operator

structural gene

terminator

 

 

 

 

ATC

3'XXXX...

TATATA...

XXXXX...

TACXXX.........XXXX........

ATT...XXX5'

 

 

 

 

ACT

5'XXXX...

ATATAT...

XXXXX...

ATGXXX.........XXXX........

...........XXX3'

enhancer -- opens up the promotor

promotor -- binding site for RNA polymerase

operator -- binding site for regulatory protein (repressor)

structural gene -- actual gene product information. On the positive strand the structural gene begins with TAC and ends with ATT, ATC or ACT. This area on the positive strand is also called the open reading frame or ORF.

terminator -- release site of the RNA polymerase

 

WHEN THE GENE IS ACTIVATED mRNA IS SYNTHESIZED
5' ->3' OFF OF THE (+) STRAND. THIS IS CALLED
TRANSCRIPTION. THE mRNA THEN IS READ AND TRANSLATED INTO PROTEIN BY THE RIBOSOMES IN A PROCESS CALLED TRANSLATION.

 

IN EUCARYOTES THERE IS OFTEN ONE MORE STEP BETWEEN TRANSCRIPTION AND TRANSLATION -- RNA PROCESSING:

many eucaryotic genes are composed of multiple coding segments (EXONS) separated by non-coding segments (INTRONS). The complete gene containing exons and introns is transcribed. However, before translation, the introns are cut out of the mRNA and the the exons are spliced together. (see pg. 197 fig. 8.8)


PROTEIN SYNTHESIS(see pgs. 198-199, fig. 8.9)

1.) Transcription

a.) DNA is copied into mRNA (or tRNA or rRNA)

b.) RNA polymerase binds to the promotor and synthesizes mRNA from the (+) strand of DNA.

c.) many copies of mRNA are made (as the first RNA polymerase moves downstream another one binds, etc.)

d.) RNA polymerase drops off when it hits the termination region

 

2.) Translation

a.) mRNA is read and used as a template to make a protein. (Need ribosome, mRNA, rRNA, tRNA and plenty of enzymes).

b.) The 5' end of the mRNA binds to the small subunit of the ribosome and then the large subunit binds to that.

c.) AUG is always the first codon read. This codon matches the anticodon on a tRNA carrying methionine. Thus methionine will always be the first amino acid of every new polypeptide.

(see pg 201, fig. 8.12 for a description of the genetic code.) Note that 61 out of 64 possible 3- letter codons specify individual amino acids. 3 of the codons do not specify amino acids -- these are call nonsense codons or stop codons.

d.) There are 61 different tRNA's. These differ in their anticodon sequence which can hybridize with the 61 codons specifying amino acids. It follows then that the different anti-codon sequences correspond to different amino acids. The amino acids are attached to the 3' end of the tRNA strand. (see pg. 200, fig. 8.10 for a description of tRNA and codon-anticodon binding.

e.) The mRNA binds to the ribosome so that the 1st codon (AUG) binds to the "P" site and the second codon is in the "A" site. The two corresponding tRNA's come in. Peptidyl transferase transfers and covalently attaches the 1st amino acid to the 2nd amino acid. The 1st tRNA leaves. The 2nd codon moves to the "P" site and the 3rd codon moves into the "A" site and the process repeats itself.

(see pg. 198-199, fig. 8.9 and also Lewis, pgs. 357-361).

f.) When a nonsense or stop codon enters the "A" site, the polypeptide is cut off of the last tRNA and both are released.

g.) In bacteria, as one ribosome begins to move down the mRNA another ribosome can bind to the promotor, and as this one moves downstream still another ribosome can bind. A piece of mRNA with multiple ribosomes bound to it is called a polyribosome. (see pg. 201, fig. 8.11 for a description of this.)

 


MUTATION

Inheritable change in the base sequence -- see Lewis pg.361, Table 16.3 for a useful analogy between language and mutation.

There are two types of mutation at the DNA level:

1.) Base Substitution / Point Mutation

2.) Frameshift / an Insertion or a Deletion

These lead to two effects at the Protein level:

1.) Missense mutation -- amino acid(s) change and the protein is still made.

2.) Nonsense mutation -- early termination of protein synthesis.

(Look at the pictures on pg. 206-207).

 

Chemical Mutagens:

Reactive Compounds -- ex.: Nitrous Acid reacts with adenine, changing it so that it "acts" like guanine, this is a A to G transition.

Base Analogs -- These "look" like the normal bases but are not. (see pg 208, fig. 8.18). Several antiviral drugs fall into this category:

Intercalating Agents -- bind tightly to DNA causing frameshift mutations. Aflatoxin is produced by Aspergillus flavus growing on peanuts and grain. This mutagen may be associated with liver cancer.

Radiation -- Ionizing radiation (gamma and beta radiation) makes reactive free radicals which react with the DNA. Ultraviolet radiation reacts with pyrimidines to form dimers. ie. thymine dimers. Many organisms (including us) have enzymes to repair UV damage. People with Xeroderma pigmentosa lack these repair enzymes and are very sensitive to sun exposure and frequntly suffer skin cancers. (see Lewis pg. 342 for a description and pictures of this disease.)


GENE REGULATION - THE LACTOSE OPERON

 

An operon is a group of structural genes which are controlled by one operator. The lactose operon (or the lac operon) is repressed by glucose and induced by lactose.

 

(See fig. 8.13, 8.14 and 8.15)

 

What is meant by glucose repression?

 

1.) When glucose is present cAMP is low.--- When glucose is absent cAMP is high

 

2.) cAMP binds to CAP - an allosteric protein, always made from the CAP gene.

cAMP binds to CAP, this opens it up and allows it to bind to the promotor. Therefore CAP only binds to the promotor when cAMP levels are high.

 

3.) RNA polymerase only binds to the promotor if CAP is bound to the promotor.

Therefore the operon cannot be transcribed unless cAMP is high; and, cAMP is only high when glucose is low. If glucose is high, CAP will not bind and the operon cannot be transcribed; ie. glucose represses transcription and expression.

 

What is meant by lactose induction?

 

1. The I protein (Inhibitor protein) is another allosteric protein. The I protein normally binds to the operator.

 

2. When the I protein is bound to the operator, the RNA polymerase cannot move downstream from the promotor. Therefore the operon cannot be transcribed if the I protein is bound to the operator.

 

3. Lactose can bind to the allosteric site on the I protein. When lactose binds to the I protein, a conformational shift occurs and the I protein falls off of the operator. This allows the RNA polymerase to move downstream and the structural genes can be transcribed. Thus, lactose can induce the transcription and expression of the operon.