Chapter 11B:Mechanisms of Microbial Genetics

5. How Prokaryotes Achieve Genetic Diversity6. Gene Regulation: the Lac Operon

4. Mutations

4. Mutations

What are Mutations?

A mutation is any change in DNA sequence:

• change of one nucleotide to another

• insertion or deletion of nucleotides or DNA fragments

• inversion or recombination of DNA fragments

What Causes Mutations?Errors in DNA replication

Chemical mutagenesis

High energy electromagnetic radiation• UV, X-rays,

gamma rays

• ~1 in every billion bps, source of spontaneous mutations

• chemicals that damage or alter nitrogenous bases

Types of MutationsSilent mutations:

• no effect on amino acid specification

Missense mutations:• change a single amino acid

Nonsense mutations:• convert a codon specifying an amino acid to a stop codon

Insertion/deletion mutations:• cause a shift in the reading frame of the gene

Silent Mutations

Point mutations that do not change the amino acid specified by a codon are referred to as “silent”.

Missense Mutations

Point mutations that change the amino acid specified by a single codon are referred to as “missense” mutations.

Nonsense Mutations

Point mutations that change a codon specifying an amino acid to a stop codon.

• Affects not only the codon with mutation, but every codon downstream is ignored, thus the polypeptide is truncated.

Frameshift Mutations

Insertions or deletions that are not multiples of 3 cause shifts in reading frame, i.e., frameshifts.

• All codons downstream are read in the wrong reading from and thus the wrong amino acids are added.

5. How Prokaryotes Achieve Genetic Diversity

Horizontal vs Vertical Gene Transfer

Vertical

Horizontal (or lateral)

• transfer to thenext generation

• transfer within thesame generation

the focus of this section

HomologousRecombination

Unless transferred DNA is circular w/Ori (plasmid),it must recombine with host DNA to be retained and passed on

Recombination can only occur between homologous (similar) DNA sequences:

• facilitated by special proteins

• original DNA is replaced and thus lost

All but 1 of the horizontal gene transfer methods we will look at

involve homologous recombination.

Methods of Gene Transfer

Bacteria can acquire DNA (i.e., new genes) in 3 basic ways:

1) Transformation• uptake and retention of external DNA molecules

2) Conjugation• direct transfer of DNA from one bacterium to another

3) Transduction• the transfer of DNA between bacteria by a virus

TransformationUnder the right conditions, bacteria can “take in” external DNA fragments (or plasmids) by transformation.

• DNA binding proteins transfer external DNA across cell envelope

• bacterial cells capable of transformation are referred to as competent

• homologous recombination can then occur

Griffith’s Transformation Experiment

1928!

Bacterial Conjugation Requires an F factor plasmid

• contains genes needed for conjugation

• directs formation of a sex pilus

Hfr Conjugation Hfr = “High

frequency of recombination

If F factor plasmid is inserted into the hostchromosome (Hfr cell), this will result in the transfer of adjacent chromosomal DNA.

• recipient can acquire donor cell genes by homologous recombination

Transduction A virus (bacteriophage) particle can transfer DNA fragments from one host cell to another followed by homologous recombination:

• requires a virus to be packaged with bacterial DNA “by mistake”

• infection of another cellby such a virus facilitatesthe gene transfer followedby homologous recombination

6. Gene Regulation

Levels of Gene RegulationThe expression of a gene into functional gene products can be regulated at multiple levels:

TRANSCRIPTION*(regulation of rate at which gene is transcribed)

mRNA transcript stability (“half-life” of transcripts)

TRANSLATION(regulation of translation of mRNA)

post-translational modifications (e.g., cleavage of polypeptides, addition of chemical groups)

*key levelof regulation

Regulation of TranscriptionThe focal point is how often RNA polymerase binds the promoter of a gene and initiates transcription which depends on:

1) Affinity of RNA polymerase for a given promoter

• some promoters are “strong” and bind RNA polymerasewith high affinity, and thus, higher frequency

• some promoters are “weak” and bind RNA polymerasewith low affinity, and thus, lower frequency

the strength of a promoter depends on its sequence

2) Influence of DNA binding proteins collectively referred to as transcription factors

• transcription factors that help RNA polymerase bind a promoterare referred to as activators or inducers

• transcription factors that inhibit or prevent RNA polymerase from binding a promoter are referred to as repressors or inhibitors

The levels and activities of various repressors & activators of transcription determine which genes are expressed, and are responsive to the cellular environment.

Let’s see how this works in genes involved with lactose catabolism in E. coli…

E. coli Growth Using Glucose

& Lactose• in the presence of both

glucose and lactose, glucose is a more efficient food source and will be used first

• when all glucose is consumed, the cells transition to using lactose as the primary food source

(lacY & lacA are not shown)

The lac operon of E. coli

The lac operon is a module of 3 genes involved in lactose catabolism, lacZ, lacY & lacA, that are transcribed in a single mRNA from a single promoter.

On either side of the promoter are 2 special sequences, the CAP site which binds the activator CAP, and the Operator which binds the lac repressor...

When Lactose is Absent:

RNA polymerase

The lac repressor protein by default is bound to the operatorsequence, thus blocking part of the promoter and preventing RNA polymerase from binding and initiating transcription of the lacZ, lacY & lacA genes.

The lac operon is OFF since there’s no need for these gene products in the absence of lactose.

When Lactose is Present Along With Glucose:

Lactose binds to the lac repressor, inducing a change in shape that prevents its binding the Operator sequence.

• with the operator no longer occupied, RNA polymerase can bind the weak promoter & initiate a low level of transcription

Since glucose (a preferred energy source) is present, the lac operon is ON “low”.

When Lactose is Present w/o Glucose:The lac repressor is inactivated by lactose, and low glucose levels trigger cyclic AMP (cAMP) production which activates CAP, a transcriptional activator, to bind the CAP site & help RNA polymerase bind the promoter.

Without glucose, lactose is a more

important food source and the lac operon is

ON “high”.

Summary of thelac operon

The lac repressorinhibits transcription

in the absence of lactose.

The CAP protein activatestranscription when glucose is unavailable and lactose

is present.

Key Terms for Chapter 11B

• transformation, transduction, conjugation, Hfr

• horizontal vs vertical gene transfer

• homologous recombination

• transcription factor, activator, repressor

• lac operon, lac repressor, operator, CAP, cAMP

• missense, nonsense, silent mutations, frame shift

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