Chapter 2: DNA and protein synthesis (C9787649)

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1 DNA structure, function

Nucleic acids are polymers (repeating structural units, see ) of nucleotides, which have three components, including:

1. Nitrogenous base, which can be either (1) adenine; (2) thymine; (3) guanine; or (4) cytosine. Adenine and guanine are 2 ring structures, called purines. Thymine and cytosine are single ring structures, called pyrimidines. This can be memorized with the mnemonic that pyrimidine has a “y”, and so does thymine and cytosine
2. Pentose sugar, a five carbon sugar, deoxyribose in DNA (hence its name deoxyribonucleic acid), and ribose in RNA (hence its name ribonucleic acid).

Deoxyribose shown  can be labeled, per the rules given , with the anomeric carbon (connected to two oxygen’s) as 1. Then going clockwise (evidently, the oxygen cannot be labeled as a carbon), there is the 2, 3 and 4 carbons. The carbon going upwards and to the left to join the $\ce{-OH}$ is the 5th carbon.

In a DNA molecule, the 5’end (the dash is called prime) (at the top of the image ) detaches its $-OH$ and connects with a phosphate group, and the other 3’ end (at the bottom of the image ) detaches its $-OH$ and connects with another phosphate group. Because the phosphate makes two bonds, this link is known as a phosphodiester bond. Therefore, the phosphodiester bond covalently bonds one deoxyribose’s 5’ end with its adjacent deoxyribose’s 3’ end [and vice versa].

Ribose [found in RNA] (shown in image ) differs from deoxyribose [found in DNA] because the 2-carbon [per the naming format used for deoxyribose] has a hydroxyl group [whereas it didn’t in deoxyribose]. This is why deoxyribose is prefixed with “deoxy”; because it lacks the oxygen found in ribose.
3. Phosphate group

 Frequently asked questions What is a nucleic acid?A polymer of nucleotides.What is a nucleotide?It consists of three things - a nitrogenous base, pentose sugar, and phosphate group.What is the difference between DNA and RNA?The pentose sugar in DNA is deoxyribose, and the pentose sugar in RNA is ribose.

A nucleoside is a nucleotide without the phosphate group, and are named after their respective nitrogenous bases, (1) adenosine, after adenine; (2) thymidine, after thymine; (3) guanosine, after guanine; and (4) cytidine, after cytosine. Nucleoside can be memorized with the mnemonic that it sounds like “suicide”, and therefore lacks a phosphate group. Additionally, if the nucleoside is derived from a DNA nucleotide (cf. RNA nucleotide), the nucleoside name should be prefixed with “deoxy”, with the exception of thymidine.

 Frequently asked questions What is a nucleosides?A nucleotide without the phosphate group. So it still has a nitrogenous base and pentose sugar.

DNA consists of two strands of nucleotides. The two pentose-phosphate backbones of the strands run in opposite directions, meaning the directions in which the 3’ [and thus 5’] carbon are facing are opposite, for example one facing up and the other facing down. [Because the strands must eventually end,] one strand is said to go from 5’ to 3’, and the other from 3’ to 5’. The 3’ end is referred to as downstream, and the 5’ end is referred to as upstream. The two strands are connected by hydrogen bonds at their adjacent nitrogenous bases, such that adenine bonds [at 2 locations] with thymine, and guanine bonds [at 3 locations] with cytosine. Because the requirement of one particular nitrogenous base to bond with a specific other [nitrogenous] base, the two strands are thus complementary. These two strands are wrapped in a twist, known as the double-helix structure.

 Frequently asked questions So DNA looks like...Two strands of nucleotides wrapped in a twist... the double helix!So there are 2 ends to the DNA. The 3' and 5'. Which is which?The lower number (3') is downstream. The higher number (5') is upstream.The two strands are connected by (what), between (what)?Connected by... hydrogen bonds. Between... adjacent bases.Not just any two bases can connect with each other, is this right?Yes. They must be complementary.Ah, so this is why the two strands run antiparallel.Exactly. Because the bases are complimentary, so they just "couldn't" run parallel - Because they are complimentary to another, not anti-compliementary to another .

Gene is a series of DNA nucleotides (i.e. a subset length of DNA) which code for a single polypeptide or RNA. One or more polypeptides make up protein. Note that in each cell, there is usually only one copy of each gene, as there is little use for keeping multiple copies. As most other copies of a cell are expected to be correct, the damaged copy can be identified by comparison to other cells, and subsequent removal as necessary. The presence only of one copy of a gene, means that mutations to a gene in a cell, can lead to loss of the related protein, and perhaps function of the cell.

 Frequently asked questions What is the relationship between a gene and DNA?A gene is a portion of the DNA, which codes for a particular thing.There is usually only one copy of each gene in a cell, but problems can be identified by...Comparison to other cells.Because there is only one copy of each gene in a cell, mutations in that gene can lead to...Loss of the related protein, and possibly function of the cell too.

2 DNA replication

Replication is the synthesis of DNA. Replication is a semiconservative process, meaning each new DNA strand consists of one old, and one newly synthesized DNA strand. Replication occurs via the replisome machine, which consists of two replicative polymerases, helicase and other machinery proteins. Replication begins at specific locations on the DNA, known as origins.

At the origin, DNA is unwound by the enzyme DNA helicase by breaking the hydrogen bonds between the two strands, creating two prongs known as a replication fork. Another enzyme DNA polymerase reads the old strands upstream (in the 3’ to 5’ direction) (both strands are read in the same direction, remembering they run in opposite direction, meaning one strand will need to be read back-to-front), and synthesizes a new strand downstream (in the 5’ to 3’ direction) (this is so that the new strand will be complementary to the old). One DNA polymerase follows DNA helicase [in front of polymerase, unzipping the DNA before polymerase acts on it], and reads and synthesizes a single continuous strand known as the leading strand. Another DNA polymerase also first reads DNA, but as noted  as it runs in the opposite direction, will need to be read back-to-front. Additionally, as the new strand is created in the opposing 5’ to 3’ direction, DNA will be synthesized in short, discontinuous segments, known as Okazaki fragments. As Okazaki fragments are discontinuous, they are adjoined by the enzyme DNA ligase, to form the lagging strand.

RNA differs from DNA apart from having ribose (cf. deoxyribose in DNA), in that it is single stranded (cf. double stranded in DNA), and replaces thymine with uracil. Note that the mnemonic that pyrimidines have a “y” thus breaks down, as uracil doesn’t contain a “y”; it does however, replace thymine. The similarity of thymine and uracil as shown  is because thymine is simply the methylation of uracil (i.e. contains an additional methyl group).

The major types of RNA include mRNA, tRNA and rRNA. rRNA is unique in that it is synthesized in the nucleolus (discussed ).

 Frequently asked questions So the difference between RNA and DNA is...Three things: RNA uses ribose [not deoxyribose]. RNA only uses a single strand [not double strand]. RNA uses thymine [not uracil].

3 Repair of DNA

Mutations are changes to the genome, not caused by normal genetic recombination such as meiosis (see ) or transposition. Transposition is where a fragment of DNA is transposed to another section.

Chromosomal aberration is where there is an abnormal number of chromosomes. For example, Down syndrome is an extra [third] copy of chromosome 21 [given there are normally only two copies of chromosome 21].

 Frequently asked questions So the gene determines everything. What can cause the gene to change?Two ways: Normal recombination. Or mutation.So Spiderman has special abilities, because he experiences a mutation?Yes.Why isn't that normal recombination?He does not have these abilities in his family tree. It spontaneously occured in just him. Mutation are random changes, that are unexpected. They are mistakes, which can turn out... to be good .

Gene mutation are changes in the sequence of a gene, and include:

• Point mutations, where a pair of nucleotides [and therefore, effectively, its base] is replaced with another nucleotide. Note that a “pair” is changed, because if only one changed, the bases will no longer correctly complement. These include:
• Silent mutation, where a codon is replaced by one which codes for the same amino acid (remembering from  that codons are degenerate, meaning several codons can code for the same amino acid), and hence has no effect
• Neutral mutation, where the amino acid is changed, but the amino acid is chemically similar, so there is no effect on protein function
• Missense mutation, where the amino acid is changed, and is chemically dissimilar, thereby causing the protein to lose its function. For example, in sickle-cell anemia, where the sixth codon of the gene for beta chain in hemoglobin has replaced glutamic acid (GAG) with valine (GUG), thereby causing RBC to lose its usual bean shape, and to adopt an abnormal and rigid sickle shape
• Nonsense mutation, where a stop codon is created in a premature location, thereby truncating the polypeptide
• Frameshift mutation, which can be caused by insertions (addition of nucleotides into the DNA) and deletions (removal of nucleotides from the DNA). As codons are read in sets of three, if a number of nucleotides not divisible by three is inserted or deleted, the reading frame will change

Wild type is the typical phenotype found in nature. Mutation from a wild type to a mutant is known as a forward mutation, and a mutation from a mutant that restores the [natural] wild type is known as a back mutation.

 Frequently asked questions So the types of mutations are...Point mutations and frameshift mutations. Point mutation is where one base set is changed. Frameshift mutation is where there is an exta [or missing] base set.When you change a point, what can happen?Something or nothing. In silent and neutral mutation is, nothing happens. In missense and nonsense mutation, something happens.What's the difference between silent and neutral mutation, then?In silent, there is a change in the codon, but the codon produces the same amino acid! So HAHA- nothing happens, couldn't trick this smart cookie . In neutral mutation, there is a different amino acid coded for, but the amino acid is not sufficiently distinct to cause any difference in protein function.What's the difference between missense and nonsense mutation, then?Missense mutation causes the classic loss of protein function. Nonsense mutation specifically produces a stop codon, thereby truncating the protein. Both are bad.

4 DNA techniques

Recombinant DNA (rDNA) is a long strand of DNA produced by laboratory methods, from shorter strands of DNA. Recombinant DNA is introduced into a bacterium, via a vector. Vector is a DNA molecule used as a vehicle to carry foreign genetic material into another cell. Viruses are commonly used as vectors. Initially, DNA polymerase (the enzyme involved with DNA replication) could not be used due to high temperatures, thereby making the process of creating rDNA time consuming, before the invention of PCR. Polymerase chain reaction (PCR) involves:

• DNA polymerase enzyme isolated from a bacterium, which is able to withstand near-boiling temperatures. Primers, which are short DNA fragments that serve as a starting point for DNA synthesis, are mixed with the rDNA
• This mixture is then heated to near-boiling temperature, causing the nucleic acid to denature. Analogous to protein denaturation [which involved breaking intermolecular, but not covalent bonds], the hydrogen bonds between complementary bases are broken, thereby separating the two strands of DNA
• The mixture is then cooled, permitting a primer to attach to the [single]DNA strand
• Because the DNA polymerase selected is optimal at a high temperature, the mixture is reheated, to permit DNA synthesis

Each time the heating-cooling cycle repeats, the amount of product is doubled (assuming maximum efficiency).

 Frequently asked questions What is recombinant DNA?DNA made from shorter strands of DNA.What is a vector?It is used to carry foreign genetic material into a cell. Because this is what viruses do, they are commonly used.What is polymerase chain reaction (PCR)?PCR is the process by which a few copies of DNA can be amplified into millions of copies. In this scenario, we are doing it to amplify the amount of rDNA we have.

5 Genetic code

Genetic code is the rules by which nucleotides are translated into amino acids [and therefore how proteins are created]. Codons are a specific reading frame of three bases. Codons code for one [and only one] particular amino acid, known as unambiguous. However, amino acids can be coded by different codons, known as degenerate. The genetic code is nearly universal throughout all life forms.

The RNA start codon is AUG, which also codes the amino acid methionine. The start and stop codons signal the start and end of a polypeptide chain respectively, and hence where translation should start and stop.

 Frequently asked questions What is genetic code?[Near] universal rules by which a codon (3 sets of bases) are translated into [specific] amino acids.What does universal means?That the code is used throughout nearly all life forms.Can a codon code for more than one amino acid?No. This is what unambiguous means. It is not ambiguous, not contentious, what each codon codes for.Can an amino acid be coded for by more than one codon?Yes. This is what degenerate means. The word "degenerate" indicates loss of some speciality. This is because by being provided with just an amino acid name, there is no idea what "specific" codon coded it.

6 Transcription

Step 1) Transcription is the first step of producing a protein from DNA. It is the synthesis of RNA (cf. replication, which was the synthesis of DNA). The distinction between "transcription" and "translation" can be memorized with the mnemonic that "transcription" contains the word "script", so is conversion from like to like (i.e. from DNA to RNA, but NOT to protein).

Transcription uses one strand of DNA as a template. DNA however, exists as two strands, to protect the DNA’s nitrogenous base from attack [because if one strand’s nitrogenous base is altered, the lack of its complement can be detected]. As RNA is synthesized from DNA, transcription must occur where there is DNA, which is in the cell nucleus, and outside the nucleus in the mitochondrial matrix.

Transcription begins when the protein transcription factors bind to a promoter. Promoter is a sequence of nucleotides that identify the start of a particular gene, and therefore where transcription can begin. Promoters are generally not intact, and the most repeated sequence of nucleotides are referred to as the consensus sequence. As the promoter is longer than the consensus sequence (since the promoter is unlikely to be intact, and therefore includes nucleotides not commonly repeated), the promoter begins before the consensus sequence. However, the closer the consensus sequence is to the promoter region, the more often transcription will occur. In prokaryotes, a promoter may be followed immediately by an operon. Analogous to replication, RNA polymerase reads DNA upstream (in 3’ to 5’ direction), and synthesizes a new stream downstream (in 5’ to 3’ direction).

Transcription [to RNA] has lower copying fidelity than replication [to DNA], as there are less proofreading mechanisms in transcription.

 Frequently asked questions What is the difference between transcription and replication [from before]?Replication is creating DNA, from DNA -> DNA. Transcription is going from DNA -> RNA.Which is better - transcription or replication?They serve different purposes. Replication is used to copy DNA. And transcription is the first part of creating a protein [discussed later]. But looking at the process alone, replication is of "higher fidelity", meaning that less errors are made, because there are more proofreading mechanisms.Where does transcription occur?Because transcription requires DNA, it occurs where DNA is located - which is primarily in the cell nucleus, and in smaller amounts in mitochondria.What is the relationship between the DNA and the RNA?RNA is copied from one strand of the DNA.What is the difference between DNA [used in transcription] and RNA polymerase [used in replication]?Whereas DNA polymerase synthesizes DNA, RNA polymerase synthesizes RNA.

Step 2) Post-transcriptional modification/control is the modification of RNA after transcription, but before the RNA leaves the cell nucleus. RNA not yet modified is known as primary RNA, or in the case of mRNA, pre-mRNA. Modification includes:

• Removal of sections of the primary RNA which do not code for protein, known as introns. The remaining sections which do code for protein, known as exons, are reconnected to form a continuous molecule
• Modification to create a start and end region for the RNA, so it can be more easily recognized, and protected from degradation

 Frequently asked questions What is post-transcriptional modification?It is a process of tidying, which also acts as a security measure to protect from damage.Where does it occur?Where the RNA is made - That is, in the cell nucleus.

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