Biochemistry Chapter Four

What is the general flow of genetic information

DNA to RNA to proteins

What is the information content of a nucleic acid

The sequence of the bases

What is each monomer of a nucleic acid made up of

A sugar, a phosphate, and a base

What is the linkage of sugars in the backbones of DNA and RNA

Sugars are linked by phosphodiester bridges between the 3’-hydroxyl of one sugar and the 5’-hydroxyl of an adjacent sugar

What carbon are the bases of a nucleotide attached to

Carbon atom 1’ in the sugar

What charge does the backbone of DNA and RNA have and what does this for the nucleic acid

The backbone has a negative charge due to the phosphate group being negatively charged. This negative charge repels nucleophilic species such as hydroxide ions that can hydrolytically cleave the phosphodiester linkages

Ester

The compound obtained when the hydrogen atom in at least one hydroxy group is an oxoacid or a hydroxoacid is replaced by an alkyl group (alkyl ester) or an aryl group (aryl ester).

Ester bond

Also called ester linkage and is the bond connecting the atom doubly bonded to oxygen and the oxygen atom bearing the alkyl or aryl group

How does the absence of a 2’-hydroxyl group in DNA increase its resistance to nucleophilic species and hydrolysis

Free hydroxide ions in solution can easily deprotonate the 2’ OH of the ribose enabling the deprotonated 2’ hydroxyl’s nucleophilic attack on the adjacent phosphorus. Phosphodiester bond is broken, cleaving the RNA backbone

Nucleoside

A base bound to a sugar

Nucleosides of DNA

deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxythymidine. By convention, since deoxythymidine rarely occurs in RNA, it is simply called thymidine in reference to DNA

The nucleosides of RNA are

Adenosine, guanosine, cytidine, and uridine

How are sugars and purine attached

The C-1’ of the sugar is attached to the N-9 of the purine by a B-glycosidic bond

How are sugars and pyrimidines attached

The C-1’ of the sugar is attached to to the N-1 of the pyrimidine by a B-glycosidic bond

What is a nucleotide in terms of a nucleoside

A nucleotide is a nucleoside with one or more phosphoryl groups attached

What are the building blocks of DNA and RNA

Nucleotide triphosphates

Mnemonic to remember the attachment of purine to sugar

PurNINE (the C-1’ of a sugar attaches to the N-9 of the purine)

Glycosidic bond

Also known as a glycosidic linkage is a type of covalent bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate

Beta position (glycosidic bond)

When the -OH is on the same side of the ring. IN the ring structure this results in an upward projection

Alpha position (glycosidic bond)

The -OH is on the opposite side of the ring. The ring structure this results in a downward projection

Nucleic acid directionality

One end has a phosphoryl group attached to the 5’ carbon of the sugar and one end has a free hydroxyl attached to the 3’ carbon of the sugar

What direction are nucleic acid sequences written in

They are written in the 5’ to 3’ direction

How long can a DNA molecule be

Pretty long, consisting of more than 1 billion nucleotides

How many nucleotides does the human genome comprise of

3 billion nucleotides per DNA strand (so 6 billion total)

How is the human genome divided

Among 23 pairs of chromosomes

What is the double helix of DNA stabilized by

Hydrogen bonds and van der Waals interactions

Hydrophobic interactions

Also called stacking forces. Interactions that help to stabilize the double helix

Semiconservative replication

Because of the base pairing rules, the sequence of one strand determines the sequence of the partner strand. The two strands can be separated and complementary sequences can be synthesized to generate two identical daughter strands. The two daughter helices have one parent strand one newly synthesized strand

Photo 51

Taken in 1951. The photo revealed that B-form DNA was a double helix with 10 nucleotide base pairs within a complete turn of the helix. The “X” in the picture indicates a helix. The dark patches indicate the bases

What distance are adjacent bases separated by and what does this mean for the structure of DNA

3\.4 angstroms at intervals of 34 angstroms which corresponds to approximately 10 nucleotides on each chain

What is the angle of rotation for each base and what does this mean for the structure of DNA

36 degrees which shows that the bases are stacked on top of one another

Base pairing rules

Account for the observation, originally made by Erwin Chargaff, in 1950, that the ratios of adenine to thymine and of guanine to cytosine are nearly the same in all species studied, whereas the adenine-to-guanine ratio varies considerably

van der Waals interactions between stacked bases

Are called stacking forces, help to stabilize the double helix

What are the different ways of depicting molecular structures

Lewis structures, condensed formulas, skeletal formula, and stereochemistry (3D arrangement), space-filling, ball-and-stick

Lewis structure

\

Skeletal formula

Condensed formula

Example: ethanol (CH3CH2OH)

Stereochemistry

Space filling molecules

A

Ball-and-stick model

B

Skeletal model

C

A form of DNA

Shorter and wider than the B form with bases at an angle rather than perpendicular to the helix axis. The sugar is in the C-3’-endo conformation

B form

The most commonly seen form of the DNA double helix. Its sugar is in the C-2’-endo configuration

Where is the A form of the DNA double helix found in

In the RNA double helices and in RNA-DNA hybrid helices, structures observed in transcription and RNA processing

What form does DNA take when it isn’t hydrated

A-DNA

Where does the C-3’ carbon atom lie in the A-form of DNA

It lies above the approximate plane defined by the four other sugar nonhydrogen atoms (called C-3’ endo)

Where does the C-2’ lie in the B-form of DNA

In the endo conformation which means that it lies out of the plane

What does C-3’-endo puckering in A-DNA lead to

A 19-degree tilting of the base pairs away from the perpendicular to the helix

Why are RNA helices induced to take the A-DNA form

Because of steric hindrance from the 2’-hydroxyl group: the 2’-oxygen atom would be too close to three atoms of the adjoining phosphoryl group and to one atom in the next base

Z-DNA

A left-handed, zigzagged form of DNA.

What is the degree of the tilt of base pairs from perpendicular to helix axis for B DNA

1 degree

\

What is the screw sense of B DNA

Right-handed

How does bacteria fit DNA into the cell without a nucleus

In order to fit inside a cell, the DNA molecule must be compacted. In E.coli the DNA double helix is a circular molecule (in terms of continuity of the DNA strands and not in reference to their geometric form) that is twisted into a superhelix by the process of supercoiling. Without coiling, E. coli chromosome, fully extended, would be about 1000 times as long as the greatest diameter of the bacterium

Stem-loop

A motif of single stranded nucleic acid which occurs when complementary sequences in the same strand form a double helix. In many cases, these double helices are made up entirely of Watson-Crick base pairs. In other cases, however, the structures include mismatched base pairs or unmatched bases that bulge out from the helix

Where do non-Watson-Crick base pairs occur frequently in terms of type of nucleic acid

They occur frequently in RNA

What do mismatched bases do in stem-loop structures of single stranded nucleic acid molecules

These mismatched bases destabilize the local structure but introduce deviations from the standard double helical structure which can be important for higher-order folding and for function

Aside form forming a stem-loop, what else can single-stranded RNA molecules do

A single-stranded RNA molecule can fold back on itself to form a complex structure. Often three or more bases interact to stabilize these structures. In such cases, hydrogen-bond donors and acceptors that do not participate in Watson-Crick base pairs participate in hydrogen bonds to form nonstandard pairings.

What can stabilize stem-loop and single stranded RNA folding

Metal ions such as magnesium ion (Mg 2+) often assist in the stabilization of these more elaborate structures

What do different, complex structures of RNA allow these molecules to do

To perform a host of functions that the double-stranded DNA molecule cannot. Indeed, the complexity of some RNA molecules rivals that of proteins, and these RNA molecules perform a number of functions that had formerly been thought to be exclusively done by proteins

Relative size of globular proteins and the mRNA that codes for it

Myoglobin mRNA is 1078 bases long but the protein (myoglobin) it codes for is only 153 amino acids long

Meselson and Stahl Semi-conservative replication experiment

Demonstrated (in 1958) that replication is semiconservative by growing bacteria in growth media supplemented with 15N. The bacteria were then shifted to growth media with 14N as the nitrogen source. Density gradient centrifugation established that upon the shift to 14N medium, newly synthesized DNA consisted of DNA with equal parts 15N-DNA and 14N-DNA, a result consistent with semiconservative replication

N14 vs N15

N15 is a heavier isotope of N14

At the start of the experiment, what would the sample of bacterial DNA look like during the Meselson and Stahl experiment?

There would be a band towards the bottom (a heavy band) indicating that the bacterial DNA was still the parent DNA with the N15 incorporated

After two generations, what would the sample of bacterial DNA look like during the Meselson and Stahl experiment

There would be some of the sample in the middle, indicating that the DNA was now in the “intermediate” expression. The DNA would have both N14 and N15 incorporated in one of the two strands each of the DNA double helix

After two generations, what would the DNA of the Meselson and Stahl experiment look like

There would be a band closer to the top which indicates that the DNA of that band consisted of two strands of the lighter N14. There would be another band in the middle indicating that there was still some DNA with the intermediate expression of the DNA with one strand with N15 and one strand with N14

How can DNA’s double helix be separated

In the laboratory, DNA strands can be separated by heating a solution of DNA, a process called denaturation or melting

Melting temperature (Tm)

The temperature at which half of the DNA molecules are denatured

How can the melting and denaturation of a DNA double helix be observed

Bases stacked in a double helix absorb less ultraviolet light than bases in a single-stranded molecule

Hypochromism

Bases stacked in a double helix absorb less ultraviolet light than bases in a single-stranded molecule which can be used to observe the denaturation of the double helix

Reannealing

After denatured DNA strands have cooled, the two strands can bind to one another to reform the double helix

Hypochromism and single-stranded DNA

Single-stranded DNA absorbs light more effectively than does double-helical DNA

Absorbance of a DNA solution at a wavelength of 260 nm

Increases when the double helix is melted into single strands

How many hydrogen bonds form a guanine and cytosine base pair

Three

How many hydrogen bonds form a adenine and thymine base pair

Two

How many proteins does DNA replication in E. coli require

More than 20, five of them being DNA polymerases

DNA polymerase

Catalyzes phosphodiester-bridge formation

Reaction of phosphodiester-bridge formation

Catalyzed by DNA polymerase:

What is required for DNA synthesis/replication

Four deoxynucleoside tripshosphates (ATP, GTP, CTP, and CTP) and Mg2+ are required, a template strand, primers, and DNA polymerase with intrinsic nuclease activity

DNA primers

From here the new DNA strand grows

What does the intrinsic nuclease activity DNA polymerases have allow them to do

Allows them to remove mismatched bases

What primes the synthesis of DNA

RNA, specifically an RNA polymerase called RNA primase

RNA primase

A RNA polymerase that synthesizes a short stretch of RNA- about five nucleotides- that is complementary to one of the template DNA strands

Why are primers needed for DNA synthesis/replication

Primer strands have a free 3’OH group which can attack the innermost phosphorus atom of the deoxyribose nucleoside triphosphate

As the phosphodiester bridge is being formed, what is being released

A pyrophosphate is released after a nucleophilic attack by the 3’OH end of the growing strand on the innermost phosphorus atom of the deoxynucleoside triphosphate. There is subsequent hydrolysis of pyrophosphate to yield two ions of orthophosphate (Pi) by pyrophosphatase which drives the polymerization forward

Retroviruses have one kind of DNA

DNA that had been transcribed from single-stranded RNA genomes by the action of reverse transcriptase

Examples of retroviruses

HIV-I

What are the genes of some viruses made of

Some viruses, such as the tobacco mosaic virus, have RNA genomes that are replicated by RNA-directed RNA polymerases

Reverse Transcriptase

Possesses several activities such as catalyzing the synthesis of a complementary DNA strand, the digestion of the RNA strand, and the subsequent synthesis of the DNA strand

Kilobase

A unit of length equal to 1000 base pairs of a double-stranded nucleic acid molecule (or 1000 bases of a single-stranded molecule).

Messenger RNA

Is the template for protein synthesis of translation

How many mRNA molecules do prokaryotes for their genes compared to eukaryotes

One mRNA molecule for a group of genes vs eukaryotes for each gene

Transfer RNA (tRNA)

Carries amino acids in an activated form to the ribosome for peptide-bond formation, in a sequence dictated by the mRNA template.Consists of about 75 nucleotides. Known as the “adaptor molecule” for protein synthesis

Ribosomal RNA (rRNA)

The major component of ribosomes. IN prokaryotes three kinds of rRNA called 23S, 16s, and 5s RNA because of their sedimentation behavior,. One molecule of each of these species of rRNA is present in each ribosome. rRNA is the actual catalyst for protein synthesis

Svedberg unit

The units for sedimentation. Bigger particles have higher S values

What is transcription catalyzed by

RNA polumerase

What does RNA polymerase have for transcription to happen

A template, activated precursors, and divalent metal ions

RNA polymerase template

The sequence of the newly-synthesized RNA is complementary to the DNA template. The DNA strand that has the same sequence as the RNA product (with T instead of U) is called the coding strand

Activated precursors

Four ribonucleoside triphosphate

Transcription reaction

DNA polymerase vs RNA poly,erase

RNA polymerase does not need primers. RNA primes the synthesis of DNA