Microbiology: The Blueprint of Life, from DNA to protein Chapter 10: Biochemistry of the Genome I. Overview A. DNA ultimately determines every aspect of a cell – from shape to function 1. DNA = __________________________ 2. Nucleotides of DNA have three units

a. A nitrogen-containing ring compound, called a ________ b. Base is covalently bonded to a 5-carbon sugar molecule, deoxyribose c. Deoxyribose is bonded to a phosphate molecule

Nucleotides – consist of a nitrogen-containing base, sugar, and phosphate

Double-Ringed Bases Purines

Single-ringed Bases Pyrimidines

Adenine

Thymine (in DNA)

Uracil (in RNA)

Guanine

Cytosine

3. __________________________ – tymine & cytosine (& uracil in RNA) – consist of a single ring 4. __________________________ – adenine and guanine – consist of two rings

5. 4 nucleotide (nitrogen containing) bases, 2 complimentary pairs a. ___________________ & ____________________ ; AT b. ___________________ & ___________________ ; GC

Complementary pairing of the nitrogen bases (image from: www.mikeblaber.org)

B. DNA encodes information to construct proteins 1. Proteins synthesized from the code of DNA are responsible for every aspect of that cell C. ________________ – the complete set of genetic information of a cell 1. _________________ – functional unit of a genome that encodes for a gene product, typically a protein D. The central dogma of molecular biology flow of information from ______________ to ___________ to protein 1. __________________________ is the process of duplicating double-stranded DNA. a. This must occur before the cell divides into 2 daughter cells. b. Each daughter cell will require a full compliment of the instructions of life.

2. __________________________ is the process of copying the information encoded on DNA into RNA. a. A set of 3 nucleotides on messenger RNA = __________________________. Codons encode for one of 20 specific amino acids 3. __________________________ is the process of interpreting the information carried by messenger RNA in order to synthesize the encoded protein. a. The aa sequence order is determined by the codons of RNA b. The _______________________________ (aa) are joined by

_________________________ to form __________________________ 1) Proteins may be structural and form membranes, etc. 2) Proteins may mediate cellular activities, e.g. Enzymes

II. Characteristics of DNA & RNA A. Characteristics of DNA 1. A single strand of DNA is a chain of nucleotides; joined by covalent bonds between the __________________________ (phosphate) group of one nucleotide and the __________________________ (hydroxyl) group of the next nucleotide 2. This creates a sugar-PO4 backbone for the strand 3. A single strand of DNA will always have a 5’ end (with PO4 at the end) & a 3’ end (with OH at the end) 4. The two strands of DNA in the double helix are __________________________;

they are oriented in opposite directions a. One strand is oriented 5’ to 3’ end (the _____________ of DNA). It

complements the strand oriented 3’ to 5’ (the ____________________ of DNA). 5. The strands are held together by weak ____________________ between

complimentary base pairing 6. The separating of double-stranded DNA is called ____________ or ____________

Nucleotides

Consist of a nitrogen-containing base, sugar, and phosphate

The sugar found in DNA is Deoxyribose. The sugar found in RNA is ribose. (images modified from: www.biologycorner.com)

1. Both are single-ring pentose (5-C) sugar. 2. The numbering of the carbon atoms runs clockwise, following organic chemistry rules. 3. Note the absence of the hydroxyl (-OH) group on the 2’ carbon in the deoxy-ribose sugar in

DNA as compared with the ribose sugar in RNA. 4. The extra -O- in the ribose backbone prevents formation of stable double-helices.

DNA

RNA

The phosphate group is shown below.

The nucleotides are assembled through dehydration synthesis. (image modified from: http://img.sparknotes.com)

The Structure of DNA DNA is a double helix molecule. The two strands of DNA are antiparallelel; one strand is oriented in the 5’ to 3’ direction, and its complement is oriented in the 3’ to 5’ direction. Hydrogen bonding occurs between the complementary base pairs; three bonds form between a G-C base pair, and two bonds form between an A-T base pair. (image modified from: http://academic.brooklyn.cuny.edu/biology)

B. Characteristics of RNA

1. __________________________ is substituted for thymine (T) 2. A __________________________ RNA fragment is transcribed from one of the two strands of DNA a. The strand of DNA that serves as the _______________ for transcription of

RNA is the _______________________ strand b. The RNA is therefore complementary to the (-) strand, its nucleotide sequence is analogous to that of the (+) strand c. The RNA transcript has the same __________________ (or polarity) as the (+) strand d. This is called __________________________ & it encodes for the aa sequence that will make a protein

Plus (+) and Minus (-) DNA & Sense RNA The DNA in the 5’ (PO4 end) to 3’ (OH end) orientation is the known as the plus (or sense) strand of DNA. The complimentary DNA strand (which is antiparallel) must be in the opposite orientation, 3’ to 5’ and is known as the minus (antisense) strand of DNA. (image modified from: http://upload.wikimedia.org)

In order to change the instructions recorded on DNA into proteins, the DNA strand must denature (unzip). The minus strand is used as a template to transcribe mRNA.

The resulting mRNA is complimentary to the minus strand of DNA. The mRNA is analogous to the plus strand of DNA. It has polarity (it is oriented in the same 5’ to 3’ direction) as the plus strand of DNA. Such mRNA is sense RNA and it encodes for the amino acid (aa) sequence of a protein.

3. There are three different functional groups of RNA molecules a. __________________________ =(mRNA) – the copy of the DNA code that bears the sequence of the aa that will be joined to from proteins b. __________________________ (rRNA) – RNA that, together with protein, forms a ribosome

1) Ribosomes are the “workbenches” on which aa will be joined to form proteins

c. __________________________ (tRNA) – type of RNA that delivers the appropriate amino acid to the ribosome during protein production (translation)

C. Regulating the Expression of Genes 1. Protein synthesis is generally controlled by regulating the __________________________ molecules. 2. mRNA is short-lived because ____________________ degrade it within minutes. III. DNA Replication A. Overview 1. DNA replication is generally __________________________ (This forms an ever- expanding “bubble” that cuts replication time in half) from a distinct starting point in circular DNA. 2. Replication of double-stranded DNA is ___________________; each of the two molecules generated contains one of the original strands (the template strand) and one newly synthesized strand. 3. The DNA chain always ________________________________

____________________________________________________; the base pairing rules determine the specific nucleotides that are added.

a. Only one nucleotide at a time is added by __________________________ b. Very accurate – 1 mistake every billion nucleotides – due to a __________________________ feature of DNA polymerase. It compares the newly synthesized (complementary) strand to the template strand as it adds nucleotides to see if they complement (if the hydrogen bonds form correctly)

Replication

Replication is a complex process in which the DNA of a chromosome is exactly copied prior to cell division. The ("parent" or template) strands of DNA in the double helix are separated (denatured) and each one is copied to produce a new ("daughter") strand. This process is said to be semi-conservative since one of each parent strand is conserved and remains intact after replication has taken place. The origin of replication is the region of a DNA molecule at which replication is initiated.

Helicase – enzyme that unwinds the DNA helix.

DNA polymerase – enzyme that synthesizes DNA using one strand as a template to generate the complementary strand.

DNA gyrase – enzyme that helps relieve the tension in DNA caused by the unwinding of the two strands of the DNA helix.

DNA can only be synthesized in the 5’ to 3’ direction, thus the DNA template can only be read in the 3’ to 5’ direction.

The leading strand is synthesized continuously.

The lagging strand is synthesized in discontinuous fragments.

4. DNA can only be synthesized in the 5’to 3’direction, thus the ____________________________________________________ ____________________________________________________

Therefore, one template strand is synthesized continuously, while one is synthesized discontinuously, requiring reinitiation & the necessity to join a series of DNA fragments

a. __________________________ – the newly forming strand of DNA that, because of its 5’ to 3’ orientation with respect to the replication fork, is synthesized continuously

b. __________________________ – the newly forming strand of DNA that must be synthesized as a series of discontinuous fragments because of its 3’to 5’orientation to the replication fork.

Replication: the Lagging Strand The lagging strand of DNA is synthesized discontinuously during replication because DNA synthesis can proceed only in the 5' to 3' direction.

Primase – enzyme that synthesizes small fragments of RNA to serve as primers for DNA polymerase during DNA synthesis.

DNA ligase – enzyme that joins the short fragments of DNA (Okazaki fragments) in the lagging strand to create a continuous complementary strand of the DNA template strand (oriented in the 5’ to 3’ direction from the origin of direction).

1. Primase initiates DNA polymerase. DNA polymerase synthesizes a complimentary strand from 5’ to 3’, until it meets the OH end of a synthesized segment of DNA.

2. Primase re-initiates DNA polymerase, which skips forward to the next segment of DNA (the enzyme reads the segment from 3’ to 5’ and synthesizes a complementary strand, adding nucleotides in the 5’ to 3’ direction). Again, the DNA polymerase will stop when it reaches the OH end of the previously synthesized fragment of DNA.

3. Primase will re-initiate DNA polymerase. And the process continues.

B. Initiation of DNA replication 1. DNA replication begins at the __________________________. a. ~250 nucleotides b. Specific proteins must recognize & bind to this site 2. Enzymes involved in DNA replication a. DNA polymerase – synthesizes DNA in the 5’ to 3’ direction, using one strand as a template to generate the complementary strand. 1) adds nucleotides to the 3’ OH group at the end of the growing chain. b. __________________________ – enzymes that “unzip” the DNA c. __________________________ – reinitiation as the replication fork proceeds forward in the lagging strand d. __________________________ – seals gaps between nucleotide fragments in lagging strand e. __________________________ – relieves tension caused by uncoiling of DNA double helix C. The Replication Fork 1. The bi-directional progression of replication around a circular DNA molecule creates __________________________. IV. Gene Expression

A. __________________________ – The process of ________________________________ 1. The enzyme __________________________ catalyzes the process of transcription, producing a single-stranded RNA molecule that is complementary and antiparallel to the DNA template. 2. In prokaryotes, a mRNA molecule can be monocistronic or polycistronic. a. __________________________ – carries 1 gene (that typically encodes for the production of one protein) b. __________________________ – carries multiple genes 3. Transcription begins when RNA polymerase recognizes and binds to a sequence of nucleotides on the DNA called a __________________________;

a. The __________________________ of the enzyme recognizes the promoter sequence

b. When transcription begins, the sigma subunit disassociates & leaves the remaining RNA polymerase (the core enzyme)

Transcription

Transcription is process in which genetic information coded in DNA is transferred into a temporary copy, known as messenger RNA (mRNA).

Promoter – nucleotide sequence to which RNA polymerase binds to initiate transcription. The promoter orients RNA polymerase so that the segment of DNA bearing the instructions for a

protein is transcribed.

Terminator – a sequence of nucleotides in DNA that, when transcribed, causes two complementary regions of the resulting mRNA to base-pair, forming a hairpin loop structure, and

causing RNA polymerase to fall off the DNA template.

4. __________________________ - RNA is synthesized in the 5’ to 3’ direction as

polymerase adds nucleotides to the 3’ OH group at end of the growing chain. a. mRNA is synthesized using the minus (-) strand of DNA (the strand oriented in the 3’to 5’direction to the promoter) as a template

5. When RNA polymerase encounters a ___________________, it falls off the DNA template and releases the newly synthesized RNA.

Transcription

RNA polymerase recognizes and binds to the promoter. The promoter orients the RNA

polymerase so that it will transcribe the strand of DNA bearing the gene.

Transcription – Elongation RNA polymerase adds nucleotides from 5’ to 3’. The mRNA molecule grows in length.

B. __________________________ – The transforming of the code of mRNA into an aa sequence of a __________________________ 1. The information encoded on mRNA (a temporary copy of genetic information) is

deciphered using the __________________________________________ a. 3 nucleotides on mRNA = _______________________. b. The codons represent one of _____________________ c. Most of the aa are represented by more than one codon, thus the genetic code is said to be ___________________

c. There is one “start” codon; _______________________ (this encodes for special version of Methionine, called f-Met. At places other than the 1st AUG after the ribosome-binding site, AUG simply codes for the aa Methionine)

e. There are __________________________; UAA, UAG, & UGA 2. A nucleotide sequence has three potential reading frames. a. mRNA carries information on where coding region actually begins

Transcription – Termination When the RNA polymerase reaches the terminator, a hairpin loop forms in the mRNA and

the RNA polymerase falls off of the chromosome.

Below are the DNA molecule, the resulting mRNA, and the freed RNA polymerase. The mRNA is now ready for the process of translation.

3. Ribosomes function as the site at which translation occurs.

a. rRNA of ribosomes identifies where to start reading mRNA b. Moves along mRNA in 5’ to 3’ direction “presenting” codons in sequential order for deciphering 4. __________________________ carry specific amino acids and act as keys that

interpret the genetic code. a. tRNA has a sequence of 3 nucleotides, the __________________________ b. The anticodon must complement the codon of mRNA in order for it to deliver its aa to the growing aa chain that will be the protein 5. Steps that occur during translation a. __________________________ of translation begins when the ribosome

Translation Initiation

1. In prokaryotes, like bacteria, translation begins even as the mRNA is still being synthesized. The 30S subunit of the ribosome binds to a sequence in the mRNA called the ribosome-binding site.

2. The first AUG (the start codon) appears after the ribosome-binding site. The first AUG

will determine the reading frame used for translation. 3. A tRNA carrying f-Met binds to the initiation complex.

a. The tRNA carrying f-Met has the anticodon UAC that complements the start codon. The tRNA is held in place by H bonds in the P-Site.

4. The 50S subunit of the ribosome joins the complex. The elongation phase then begins.

binds to the ribosome-binding site on the mRNA molecule; this occurs even while the mRNA is still being synthesized.

1) Translation starts at the first AUG downstream of that site. 2) The ribosome moves along mRNA in the 5’ to 3’ direction so that one codon is translated at a time.

b. __________________________ – the 70S ribosome has 2 sites to which tRNA-carrying aa can bind, the __________ (peptidyl site) and the __________________ (acceptor site)

1) The ribosome will present the “start” codon AUG at the P-site 2) The initiating tRNA, carrying f-Met, (with the anticodon UAC) binds to the P-site 3) A tRNA that recognizes the next codon on the mRNA fills the unoccupied A-site 4) f-Met is then covalently bonded to the aa carried by the tRNA that has entered the A-site (this bond is called a peptide bond) a) This transfers the f-Met from the initiating tRNA to the aa carried

by the incoming tRNA c. __________________________ – the ribosome advances a distance of one

codon 1) The tRNA that carried the f-Met is released through an adjacent site,

the ____________ (exit site) 2) The remaining tRNA (which now carries the 2-aa chain) occupies the P-site 3) The A-site is temporarily vacant 4) A tRNA that recognizes the next codon quickly fills the empty A-site 5) The process repeats d. __________________________ - Translation terminates when the ribosome reaches a stop codon. 1) Stop codon – a codon that does not code for an aa & is not recognized

by a tRNA 6. Proteins are often modified after they are synthesized; those that contain a signal

sequence are transported to the outside of the cell.

Translation - Elongation 1. A tRNA that recognizes the codon in the A-Site hydrogen bonds to it.

a. This tRNA is carrying the amino acid (aa) Proline. 2. F-Met covalently bonds to Proline (this is the peptide bond). This frees the initiation tRNA.

3. Translocation – the ribosome advances one codon. The initiating tRNA moves to the E-Site and will now

exit (it will go into the cytoplasm and attach to another Methionine amino acid. a. The tRNA that was in the A-Site moves to the P-Site. b. The A-Site is temporarily vacant until it is filled with a tRNA (carrying Tyrosine) that complements

the codon presenting in the A-Site.

4. A peptide bond forms between the aa carried by the tRNA on the P-Site and the aa carried by the tRNA on

the A-Site. This frees the aa on the P-Site. a. The ribosome advances one codon.

5. A tRNA that complements the codon in the newly empty A-Site now fills that site. The tRNA in the E-Site now exits.

6. A peptide bond forms between the aa carried by the tRNA in the P-Site and the aa carried

by the tRNA in the A-Site. This frees the aa in the P-Site. 7. The process continues, with the ribosome advancing one codon at a time, until the “stop

codon”.

V. Principles of Regulating Gene Expression – cells must cope with changing conditions. It is most E efficient to control what is synthesized A. Genes encoding _______________________________ are always active & these

enzymes are synthesized at the same levels all the time

B. Genes encoding enzymes that can be _______________________ are turned on only by certain conditions; those that can be __________________________ are turned off by certain conditions.

Translation - Termination

1. Elongation ends when the ribosome reaches the stop codon, a codon that does not code for an amino acid and is not recognized by a tRNA.

2. The newly synthesized polypeptide (protein) is freed. 3. The ribosome falls off and dissociates.

Using what you have learned, please fill in the missing information:

mRNA codon AUG CCG UAC GAG AUU CUG AAA GCU UUU CAG UAA

tRNA anticodon UAC GGC AUG CUC

Amino Acid f-Met Pro Tyr Glu

Image from: http://biology.kenyon.edu/courses

VI. Mechanisms to Control Transcription A. DNA Binding Proteins 1. Many genes have a __________________________ near their promoter to which a specific protein can bind, controlling transcription. It acts as an “on/off” switch. 2. A __________________________ is a regulatory protein that blocks transcription.

a. One Mechanism – repressor must bind to a molecule, the __________________________, to change its shape & it then blocks transcription

ex) Tryptophan production b. Another Mechanism – Repressor is in a shape that stops transcription.

However, it can bind to an __________________________ that causes it to be unable to stop transcription

ex) Lactase enzyme production 3. An __________________________ is a regulatory protein that enhances transcription. a. In this case, the gene has an _____________________ proceeded by an

activator-binding site ex) Maltase enzyme production

B. Alternative Sigma Factors – simultaneous activation of multiple genes 1. Alternative sigma factors direct RNA polymerase to recognize specific promoter sequences that are needed only under specific environmental conditions such as __________________________ 2. they are a common mechanism of global regulation. VI. Modulating Expression in Response to Environmental Conditions A. ____________________________________________________ 1. The product of a biosynthetic pathway, for example tryptophan, regulates the genes that encode the enzymes required for its synthesis by functioning as a co-repressor. B. ____________________________________________________ 1. The substrate of a degradative pathway regulates the genes that encode the enzymes required for its degradation by serving as an inducer. 2. Lactose activates the lac operon by binding to a repressor. This prevents the repressor from blocking transcription. 3. Maltose activates the genes required for its degradation by binding to an activator. This allows the activator to bind to the activator-binding site. C. ____________________________________________________ 1. Catabolite repression turns off certain genes when more readily degradable energy sources such as glucose are available. a. ↑ glucose ↓ cAMP – no transcription of enzymes to degrade other sugars

b. ↓ glucose ↑ cAMP – transcribe genes to create enzymes to degrade other sugars

D. ____________________________________________________ – transmits information from outside (about osmotic pressure, the concentration of cells, and N availability, etc.) to inside the cell to respond to changes in environmental conditions

1. Two-component regulatory systems utilize a ____________ that recognizes

changes outside the cell and then transmits that information to a response __________________________

a. Sensor – 2 proteins that span cytoplasmic membrane b. A change in the environment modifies a region on the internal portion of the sensor. 1) e.g. Phosphorylating a specific aa. The phosphoryl group is

transferred to a response regulator. The modified regulator acts as an activator or repressor.

2. Bacteria that utilize __________________________________ synthesize a soluble compound, a homoserine lactone, which can move freely in and out of a cell. Only when that compound reaches a critical concentration does it activate specific genes.

a. Marine bacterium Vibrio fischeri can emit light at critical mass. E. Natural selection 1. The expression of some genes changes randomly, presumably enhancing the chances of survival of at least a subset of a population under certain environmental conditions. 2. ________________________________________________ is a routine change in

the expression of surface proteins such as flagella, pili, and outer membrane proteins. a. Stays ahead of our body’s defenses. E.g. Neisseria gonorrhoreae 3. _____________________________________________ is the routine switching on and off of certain genes. VII. Genomics A. Analyzing a DNA Sequence

1. When analyzing a DNA sequence, the nucleotide sequence of the __________________________ is used to infer information carried by the corresponding RNA transcript. a. Start codon, AUG, is used to determine the (+) strand 2. Computers are used to search for open reading frames (ORFs). Read from a start codon to a stop codon. 3. The nucleotide sequence of the ORF or the amino acid sequence of the encoded protein can be compared to other known sequences by searching a computerized database that contains all published sequences.

B. The E. coli Genome 1. In ___________, the ______________________ base-pair genome of E. coli, which has served as a model bacterium in many scientific studies, was published.