Microbiology: From DNA to Protein

Structure of DNA and RNA

DNA is a double-stranded, antiparallel double helix, with a sugar phosphate backbone

• used for long-term genetic storage, while

RNA is typically a single-stranded molecule

• used as a temporary working copy for protein synthesis

Sugars and bases associated with DNA and RNA

DNA: Deoxyribose and Adenine, Thymine, Guanine, Cytosine

RNA: Ribose and Adenine, Uracil, Guanine, Cytosine

Be able to distinguish the 5’ end from the 3’ end

• 5’ End: This end has a free phosphate group attached to the 5’ carbon of the terminal sugar ring.

• It does not link to another nucleotide upstream.

• 3’ End: This end has a free hydroxyl group attached to the 3’ carbon of the terminal sugar ring.

• New nucleotides attach to this hydroxyl group during DNA synthesis

Which bases form complementary pairs and the orientation of strands in a double stranded molecule?

adenine pairs with thymine, and guanine pairs with cytosine

two strands run in opposite, antiparallel orientations

• one runs 5' to 3' while the other runs 3' to 5'

What is the structure of the prokaryotic chromosome? How does this compare to the

structure of the eukaryotic chromosome?

Prokaryotic: Single double stranded, closed, and circular

Eukaryotic: Multiple double stranded, linear

What processes are involved in gene expression?

DNA Replication: Duplicates DNA molecule so its encoded information can be passed onto next generation

Transcription: Copies the information in DNA into RNA

Translation: Interprets the information carried by RNA to synthesize the encoded protein

What is the central dogma?

Flow of Information

DNA —→ RNA (transcription)

RNA—→ Protein (translation

What are the possible products of gene expression?

a functional protein or a functional RNA molecule

What functions are associated with RNA?

to convert the genetic code stored in DNA into functional proteins that drive cellular life

• Messenger RNA aka transcript: Copies genetic instructions from DNA and carries code to ribosomes for protein synthesis

• Ribosomal RNA: Combines with proteins to form ribosomes to help link amino acids

• Transfer RNA: matches mRNA codons with their specific amino acids

What is meant when we say that DNA is replicated in a semi-conservative manner?

It means that each of the two molecules created contains one original strand paired with a newly synthesized strand

What is the role of a Primer?

What type of nucleic acid is used and why is it necessary?

Fragment of nucleic acid to which DNA polymerase can add nucleotides only to a existing fragment
RNA is the nucleic acid used it is necessary because:

• DNA polymerase: is unable to start a new strand from scratch. It can only attach new nucleotides to a pre-existing chain

DNA gyrase: function and why is it necessary?

Enzyme that temporarily breaks the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix

DNA ligase: function

Enzyme that joins two DNA fragments together by forming a covalent bond between the sugar and phosphate residues of adjacent nucleotides

DNA polymerase: what needs to be present in order for DNA polymerase to

synthesize DNA

A single-stranded DNA molecule that guides the enzyme on which complementary nucleotides to add

An RNA primer that is base paired to the template strand to provide a starting point

What is the role of DNA polymerase I and DNA polymerase III

DNA Polymerase III: It rapidly matches and attaches new DNA nucleotides to template strand at 3’ end of one Okazaki fragment and then encounters the 5’ end of another

DNA Polymerase I: It removes the temporary RNA primers and replaces them with DNA

What is the direction of DNA synthesis?

What is the direction that the template strand is read?

Direction of DNA synthesis: 5' to 3' (new nucleotides are added only to the 3' end of the growing strand).

Direction the template strand is read: 3' to 5' (DNA polymerase moves from the 3' end toward the 5' end)

Okazaki fragment: what is it and is it found on the leading or lagging strand

Nucleic acid fragment produced during discontinuous synthesis of the lagging strand of DNA

Origin of replication: what is it and what events take place here

Distinct region of a DNA molecule at which replication is initiated and then proceeds in both directions

Opening the strands creates an open region known as a replication bubble

This creates replication forks where synthesis occurs

Primase: function

Enzyme that synthesizes small fragments of RNA to serve as primers for DNA synthesis

Be able to list the steps involved in DNA replication in prokaryotes in the order in which

they occur.

Be sure to include the enzymes involved at each step.

1. A DNA helicase unzips the DNA helix and separates the strands at a replication fork

2. DNA gyrase breaks the strands of DNA at a replication fork and synthesis of leading strand continues

3. Single stranded DNA binding proteins stabilize the strands

4. Primase synthesizes RNA primers

5. DNA Polymerase III continuously synthesizes the leading strand in the 5' to 3' direction toward the replication fork. It also synthesizes the lagging strand away from the fork in short Okazaki fragments.

6. DNA Polymerase I removes the RNA primers and fills in the gaps with DNA nucleotides.

7. DNA Ligase seals the nicks between Okazaki fragments to create a continuous strand.

What processes are involved in gene expression?

Transcription and Translation

What are the products of gene expression?

mRNA, tRNA, rRNA and proteins

What are the basic components of a gene?

What functions are associated with these components?

DNA and genetic info to make RNA product

Coding region: region that is transcribed

• can be monocistronic (one region) or polycistronic (multiple regions)

What is the function of the Template strand (-) in transcription?

What about the non-template strand (+)?

Template: Strand of DNA that serves as the template for RNA synthesis

• resulting RNA is complementary to this strand

Non-template: Strand of DNA that is complementary to the template strand

What is the function of the Promoter in transcription?

Nucleotide sequence to which RNA polymerase binds to initiate transcription

Starts at 3’ end of template strand

What is the function of RNA polymerase in transcription?

Enzyme that synthesizes RNA using one strand of DNA as a template

What is the function of the Sigma factor in transcription?

Component of RNA polymerase that recognizes a promoter.

Cells can have different types of sigma factors that recognize different promoters

• This allows the cell to transcribe specialized sets of genes

What is the function of the terminator in transcription?

Nucleotide sequence at which RNA synthesis stops

RNA polymerase detaches from DNA template and releases the newly synthesized RNA

What is the direction of synthesis of RNA during transcription?

In what direction is the template strand read?

Synthesis is 5’ to 3’

Template strand is read 3’-5’

Steps in Transcription!!!

1. Initiation: RNA polymerase binds to the promoter and melts a short stretch of DNA

2. Elongation: Sigma factor separates from RNA polymerase, leaves core enzyme to complete transcription.

• RNA synthesized 5’ to 3’ as nucleotides are added to the 3’ end

• reannealing strands come together

3. Termination: When RNA polymerase encounters a terminator, it detaches from the DNA and releases the new synthesized strand of RNA (NOT A STOP CODON)

What is the general structure of a mRNA molecule?

single-stranded linear nucleic acid composed of a specific sequence of ribonucleotides (adenine, cytosine, guanine, and uracil) organized into distinct functional and structural regions

What is the coding region and what defines the beginning and end of the coding region?

The region that is translated into specific amino acid sequences that encode a protein

A start and stop codon define beginning and end

What are the 5’ and 3’ UTR’s on a mRNA?

They are the untranslated regions of the mRNA strand

What occurs, in general, during the process of translation?

Conversion of a RNA base sequence into a sequence of amino acids that eventually forma protein

What is the product of translation?

a sequence of amino acids that folds into a functional protein

Are all RNA molecules produced during transcription translated into proteins?

No!! messenger RNA is translated into proteins

Transcription produces a wide variety of non-coding RNAs that function directly as RNA molecules without ever becoming proteins:

• rRNA forms ribosomes

• tRNA carries amino acids to the ribosome

What is the function of an Anticodon in translation?

sequence of three nucleotides in a tRNA molecule that is complementary to a particular codon in mRNA

allows tRNA to recognize and bind to the appropriate codon

What is the function of mRNA in translation?

The type of RNA molecule that contains the genetic information decodes during translation

what is the reading frame and how is it established?

Stretch of nucleotide grouped into sequential triplets that code for amino acids

an mRNA molecule has three potential reading frames but only one is used

What the function of the ribosome?

Structure that facilitates the joining of amino acids during the process of translation

Composed of protein and rRNA

What is the structure of prokaryotic and eukaryotic ribosomes?

Prokaryotic: 70S= 50S + 30S subunits

Eukaryotic: 80S= 40S + 60S subunits

What are E, A, and P sites in the ribosome?

A Site (Acceptor): The incoming tRNA carrying a new amino acid lands here to check if it matches the mRNA code.

P Site (Peptidyl): It holds the tRNA that is attached to the growing chain of amino acids.

E Site (Exit): The empty tRNA moves here after giving away its amino acid, and then leaves the ribosome.

what is the ribosome binding site and where is it located?

sequence of nucleotides in mRNA to which a ribosome binds

It is located approximately 6 to 10 nucleotides upstream of the AUG start codon in the 5' untranslated region of the mRNA.

What is the function of rRNA in translation?

The type of RNA molecule present in ribosomes

What is the start codon in translation and what is the function?

Methionine: AUG

Codon at which translation is initiated

First AUG after the ribosome binding site

What is the stop codon in translation and what is the function?

Three stop codons: UAA, UAG, UGA

Codon that terminates translation, signaling the end of the protein

What is tRNA in translation and what end does the amino acid attach to?

Type of RNA molecule involved in interpreting the genetic code during translation

Each tRNA molecule carries a specific amino acid dictated by its anticodon

The amino acid attaches to the 3' end of the tRNA molecule

In what orientation does the anticodon pair with the codon?

Complementary

Anticodon: 3’ to 5’

Codon: 5’ to 3’

What is a codon?

A sequence of three consecutive nucleotides in a DNA or RNA molecule that forms a unit of genetic information

Encodes a specific amino acid or signals the start or end of protein synthesis

What is meant by the coupling of transcription and translation?

While transcription is still in progress, ribosomes can start translation before transcription is finished.

In what organisms does this coupling occur?

prokaryotes

Why does coupling occur in prokaryotes but not eukaryotes?

Prokaryotes:

They do not have a nucleus. Everything floats together in one room (the cytoplasm).

Done at once: As soon as the cell starts reading DNA to make a message (transcription), ribosomes jump right onto that message to build proteins (translation).

Eukaryotes:

• They have a nucleus that acts like a private room for DNA.

• Done in steps: The cell copies the DNA into a message, the message is cleaned up and edited, the message leaves the nucleus, and then ribosomes read it to build proteins.

Describe the process of translation initiation.

The initiating tRNA carrying the amino acid f-met, base pairs with the start codon and occupies the P site.

Describe the sequence of events that occur during the elongation portion of translation.

1. A charged tRNA that recognizes the codon in the previously empty A-site now fills that site

2. The ribosome catalyzes the joining of the amino acid carried by the tRNA in the P-site to the one carried by the tRNA in A-site (peptidyl transferase)

3. Translocation: ribosome moves forward on the 3’ end one codon length

4. Repeat this process advancing along the mRNA

Describe what happens during translation termination.

Translation terminates when the ribosome reaches a stop codon

No tRNA molecules recognize a stop codon

Components disassemble releasing a newly formed polypeptide

What events occur after translation is completed in prokaryotes?

Protein folding

• Chaperone proteins assist with folding

Protein modification

• signal sequences tell cell what needs to be modified

What events occur during eukaryotic mRNA processing?

1. Eukaryotic DNA contains introns which interrupt coding regions (exons)

2. Transcription generates pre-mRNA that contains introns, A cap and poly A tail are added

3. splicing removes introns to create functional mRNA

4. mRNA is transported out of nucleus to be translated in the cytoplasm

What is mRNA splicing and why does it occur?

It removes introns to create functional mRNA

Describe how antigenic variation and phase variation affect natural selection.

Antigenic variation: random expression of one of several genes for a specific protein

• tries to get around the immune response

Phase variation: routine switching on and off of genes

What is gene regulation and why is gene regulation needed?

Not all genes need to be expressed at all times and helps the cells not waste cellular resources

What is the difference in the regulation of constitutive, inducible, and repressible enzymes?

Constitutive: continuously synthesized

Inducible: only synthesized under certain conditions; when substrate is present

• normally off —→ turns on

Repressible: routinely synthesized, but can be turned off; too much gene product

• normally on —→ turns off