Bio 130, November 17, 2000
Chapter 17:  Translation and Signal Sequence
Chapter 18:   Microbial Models:  The Genetics of Bacteria and Viruses

·        Elongation Fig. 17.16

§         tRNA then enters A site,

§         Requires elongation factor and GTP hydrolysis

§         Peptide bond formation catalyzed by ribosome (by ribozyme)

§         Joins polypeptide extended from tRNA in P site to the amino acid carried by the tRNA in the A site; transfer polypeptide from tRNA in P site to tRNA in A site

§         tRNA in A site is translocated to P site; empty tRNA in P site translocated to E site, then exits the ribosome

§         mRNA moves through the ribosome in one direction

§         Energy supplied by GTP hydrolysis

·        Termination Fig. 17.17

Signal peptides

·        All protein synthesis begins in cytoplasm on free ribosome

·        If newly synthesized peptide contains a signal peptide, it cues the ribosome to attach to the ER

·        signal peptide is sequence of ~20 amino acids, located at or near the amino terminal portion of the polypeptide

·        Signal peptide is recognized by a signal-recognition particle (SRP) as it emerges from the ribosome

·        SRP is a protein-RNA complex

·        SRP acts as an adapter that brings ribosome to receptor protein on ER membrane

·        Signal peptide usually cleaved by enzyme

Chapter 18: Microbial Models:  The Genetics of Bacteria and Viruses

What is a virus?  Figures 18.1 and 18.2

·        Infectious particles consisting of nucleic acid (genome) enclosed in a protein coat (capsid), and in some cases, a membranous envelope

·        Very small in size

·        Some types infect bacteria (=bacteriophage)

·        Some types infect plant cells

·        Animal viruses

·        Isolated virus=virus particle=virion

Viral genomes (set of genes)

·        Unusual in that genome can be double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA

·        DNA genome=DNA viruses

·        RNA genome=RNA viruses

·        Usually single linear or circular molecule of nucleic acid

Capsids

·        Protein shell that encloses viral genome

·        Different shapes

·        Many are easily crystallized

Viral envelopes

·        Found in many animal viruses

·        Membranes surrounding capsid

·        Derived from host cell, but contain additional virally encoded proteins

Viral reproduction:  Figure 18.3

·        Viruses are obligate intracellular parasites:  they reproduce only within a host cell

·        A virus can only infect a limited range/type of host cell

·        Animal viruses are usually tissue or cell-type specific; that is they will only infect one particular type of cell

·        Steps of viral life cycle

o        Virus binds to host cell

o        Lock and key fit between viral protein and host cell surface receptor

o        Viral genome, via variety of mechanisms, enters cell

o        Viral genome "commandeers" its host, using host cells machinery to copy the viral genome and synthesize viral proteins

·        DNA viruses usually use host cell's DNA polymerases

·        RNA viruses must use special virally encoded polymerases (such as reverse transcriptase)

Bacteriophages

·        Some double stranded DNA viruses can reproduce by two alternative means

·        Lytic cycle Figure 18.4

o        Phage infects cell, reproduces, and is released by lysis of host cell

o        Lytic viruses only reproduce by lytic cycle

o        T4 is a lytic virus

·        Lysogenic cycle Figure 18.5

o        Replicates viral genome without killing cell

o        Viruses with lysogenic cycles called temperate viruses

o        λ phage

o        In lysogenic cycle, viral genome is in incorporated into host cell's genome by crossing over at a specific site

o        Viral genome is then called a prophage

o        As host cell replicates, viral genome is replicated

o        At some point, viral genome is activated and initiates lytic cycle

Animal viruses

·        Very diverse, many modes of reproduction Table 18.1

·        Viral envelopes Figure 18.6

o        Outer membrane found on many animal viruses

o        Helps virus enter the host cell

o        Typically a lipid bilayer (derived from host cell plasma membrane) with virally encoded proteins protruding from it

o        Virally encoded proteins typically important for binding and entering host cell

·        Herpesvirus envelope derived from nuclear envelope

o        Herpesvirus genome integrated into host cell DNA (as provirus)

o        Virus usually remains latent

o        Stress causes virus to become active, provirus leaves genome and intiates viral production

o        Blisters form as a result

RNA viruses

·        Some phages, most plant viruses, and many animals viruses have RNA as genetic material

·        RNA is used as genetic material in many different ways

o        Functions directly as mRNA

o        Functions as template for mRNA

o        Functions as template for DNA copy of genome (retroviruses)

o        In retroviruses, information flow is:  RNA -> DNA -> RNA -> protein

§         Reverse transcriptase uses RNA as template for DNA synthesis

§         HIV life cycle Figure 18.7

Viroids and prions

·        Viroids are small molecules of naked RNA that infect plants

·        Prions are infectious proteins

o        Cause a number of degenerative brain diseases

o        Mad cow disease

o        Scrapie in sheep

o        Creutzfeldt-Jakob disease in humans

o        Maybe a misfolded version of a normal protein, which redirects protein folding in infected cells, generating more misfolded proteins which can infect other cells Figure 18.9

Bacteria

Escherichia coli (aka E. coli) are the "laboratory mice" of molecular biology

Bacteria are very adaptable to their environments

·        Bacteria divide by binary fusion, preceded by replication of bacterial chromosome Fig. 18.10

·        Bacteria proliferate rapidly (20 minutes for E. coli)

·        Cannot increase genetic diversity by meiosis and fertilization

·        Low mutation rate, but mutations can have a significant effect on genetic diversity because of rapid rate of proliferation of E. coli

·        Genetic recombination also adds genetic diversity to a population

Genetic recombination can produce new bacterial strains

·        Genetic recombination for bacteria = combining DNA from two individuals into a single individual

·        Example of arg+trp- and arg-trp+ strains interacting to produce arg+trp+ strain Fig. 18.11

·        Genetic recombination can occur by

o        Transformation

o        Transduction

o        Conjugation

Transformation

·        You did this in your last lab

·        Alteration of a bacteria's genotype by the uptake of a naked, foreign piece of DNA

·        Foreign DNA can be incorporated into bacteria's chromosome by crossing over at homologous (similar) sequences

·        If foreign DNA is plasmid DNA, does not incorporate into bacterial chromosome

Transduction

·        Phages carry DNA from one bacteria to another

·        Generalized transduction:  DNA from infected cell is accidentally packaged into a phage, and transferred to another bacteria by infection;

o        Occurs in a lytic cycle Fig. 18.12

·        Specialized transduction:  when prophage (of lysogenic cycle) is excised from bacterial genome, it takes a small amount of the bacterial DNA with it.  This DNA is then packaged into a phage and transferred to another bacteria by infection

Conjugation

·        Conjugation is the direct transfer of DNA between bacterial cells that are temporarily joined

·        "Maleness" is conferred by a piece of DNA called an F factor

o        F factor can exist in bacterial chromosome or as a plasmid

o        F factor encodes genes required for formation of sex pili

o        bacteria with F factor are F+

·        "Female" cells are F-

·        "Male" bacteria form sex pili which attaches to "female" bacteria Fig. 18.13

·        Copy of F factor is transferred across pili Fig. 18.14

o        if plasmid F factor, plasmid is transferred

o        if F factor is integrated into the bacterial chromosome, bacterial DNA is also transferred

·        If F factor integrates into bacterial chromosome, bacteria is said to be an Hfr cell (for high frequency of recombination)

Mobile genetic elements

·        Transposon is a piece of DNA that can move from one point to another in bacterial cell's genome

·        Transposons do not exist as independent genetic elements (like plasmids)

·        Can move DNA within bacterial chromosome or from one plasmid to another (i.e. to give multiple drug resistance)

·        Some transposons "jump" from one location to another; cut and paste transposition

·        In replicative transposition, the transposon is copied, and the copy inserts in the new location

·        Insertion sequences Fig. 18.15

o        Simplest transposons

o        Contain one gene, transposase, which catalyzes transposition

o        Ends are inverted repeats of 20-40 base pairs

·        Mechanism shown in Fig. 18.16

o        DNA is cut in staggered fashion by transposase

o        Insertion sequence inserted (also by transposase)

o        DNA polymerase and ligase fill in DNA and ligate ends

o        DNA next to insertion sequence thus contains direct repeats

·        Composite transposons contain additional genes Fig. 18.17

o        Additional genes are "sandwiched" between two insertion sequences that travel together

Control of bacterial gene expression

·        Two levels of metabolic control Fig. 18.18

o        Cells can vary the numbers of specific enzyme molecules made

o        Regulate gene expression

·        Cells can adjust the activity of the enzymes already present

o        Occurs quickly, as it does not require transcription

·        Tryptophan example:  if cell is growing in presence of tryptophan, it does not need to synthesize it

o        When tryptophan is present, it inhibits the first enzyme involved in synthesizing tryptophan

o        The presence of tryptophan also causes cell to stop making the enzymes needed for tryptophan synthesis

§         Occurs at the level of transcription

Operons

·        In bacteria, the genes for a particular pathway/metabolic process are clustered together on the chromosome, in a unit called an operon

·        A single promoter serves all the genes of the operon

·        The clustered genes constitute a transcription unit- one long mRNA is made

o        This long mRNA is translated into separate polypeptides because the mRNA contains separate start and stop codons for each

·        An operon has a single on-off switch that controls the expression of all the genes

o        This switch is termed an operator

o        Operator is located within the promoter or between the promoter and the genes

o        Controls access of RNA polymerase to the genes

·        This cluster of genes, promoter and operator is termed an operon

·        The operon can be switched off by a protein called a repressor

o        Repressor is the product of a regulatory gene, which is not part of the operon and has its own promoter

·        Regulatory genes are transcribed continuously at a low rate

·        Many repressors are allosteric molecules, with two shapes:  active and inactive

·        Corepressor is a small molecule that interacts with a repressor to switch an operon off

The trp operon

·        trp operon is an example of a repressible operon Figure 18.19  (know this for the exam)

·        trp operon is normally transcribed

·        When tryptophan is present, it binds with the trp repressor, triggering an allosteric change

·        The trp repressor with bound tryptophan binds to the operator, shutting off transcription of the trp operon

·        Tryptophan is a corepressor

 

Repressible versus inducible operons

·        trp operon is repressible because transcription is inhibited by a specific small molecule (i.e. tryptophan) interacts with a regulatory protein

·        Inducible operons are stimulated when a specific small molecule interacts with a regulatory protein

·        lac operon is an example of an inducible operon Figure 18.20  (know this for the exam)

·        lac operon encodes enzymes needed for metabolism of lactose (milk sugar)

·        lacI is the regulatory gene; it encodes an allosteric repressor that binds to the operator in the absence of lactose

·        When lactose is present, an isomer, allolactose, binds to the repressor and causes a conformational change so that the repressor can no longer bind to the operator

o        The lac operon is then transcribed

·        Allolactose is an inducer, as it induces transcription of the operon

·        Also an example of negative control, because the operon is turned off by the repressor

Figure 18.20

Figure 18.21

 

Positive gene regulation  Figure 18.21

·        Transcription of lac operon requires both that lactose be present and that glucose be in short supply

·        If glucose levels are high, cell does not need to synthesize enzymes to catabolize glucose

·        Glucose levels detected by interaction of an allosteric protein with a small organic molecule (be familiar with this for the exam)

o        cyclic AMP accumulates when glucose is absent

o        cAMP receptor protein (CRP) binds cAMP, and is an activator of transcription

·        CRP binding site next to promoter of lac operon

o        CRP plus cAMP bind to this site, making it easier for RNA polymerase to bind to the promoter and start transcription