Storing and using genetic information

Question 1

Describe the double-helical structure of DNA.

Answer 1

Summary of the Double-Helical Structure
Two strands of nucleotides running antiparallel.
The strands are made of a sugar-phosphate backbone.
Hydrogen bonds between complementary nitrogenous bases (A-T, C-G) hold the strands together.
The molecule twists into a right-handed helix with major and minor grooves.
The structure is stable and allows for the precise replication and transmission of genetic information.
DNA can be further compacted and packaged into chromosomes through supercoiling and wrapping around histones.
This double-helical structure is crucial for maintaining genetic integrity, controlling gene expression, and enabling the complex processes of DNA replication, repair, and transcription.

Question 2

Describe the process by which DNA is replicated.

Answer 2

DNA Replication: An Overview
DNA replication is the process by which a cell makes an identical copy of its DNA. This process is essential for cell division, whether in mitosis (for somatic cells) or meiosis (for gametes). DNA replication ensures that each daughter cell receives a complete set of genetic instructions. The process is highly accurate and tightly regulated to maintain genetic integrity.

DNA replication occurs in three major stages:

Initiation
Elongation
Termination
Each of these stages involves a coordinated series of molecular events, and several key enzymes and proteins are involved in the process.

1. Initiation of DNA Replication
   Origin of Replication:

DNA replication begins at specific sites on the DNA molecule called origins of replication. In eukaryotes, there are many origins along the chromosomes, whereas in prokaryotes, there is typically a single origin.
Helicase and Unwinding the DNA:

The enzyme DNA helicase unwinds the double helix by breaking the hydrogen bonds between complementary base pairs (A-T, C-G), creating two single-stranded DNA (ssDNA) templates.
As the helicase moves along the DNA, it creates a replication bubble with two replication forks at either end, where the DNA is actively being unwound.
Single-Strand Binding Proteins (SSBs):

After the DNA strands are separated, single-strand binding proteins (SSBs) bind to the exposed single-stranded DNA to prevent the strands from reannealing (coming back together) or being degraded.
Topoisomerase:

As the helicase unwinds the DNA, it causes supercoiling ahead of the replication fork. To alleviate this strain, topoisomerase enzymes (such as DNA gyrase in prokaryotes) make temporary cuts in the DNA to relieve the tension, then rejoin the DNA strands.
2. Elongation (Synthesis of New DNA Strands)
Primase and RNA Primer:

DNA replication cannot start from scratch; it requires an RNA primer to provide a free 3’ hydroxyl group (-OH) for the addition of nucleotides.
The enzyme primase synthesizes a short RNA primer (typically 5-10 nucleotides long) complementary to the single-stranded DNA template. This primer serves as the starting point for DNA synthesis.
DNA Polymerase III (in prokaryotes) / DNA Polymerase α (in eukaryotes):

DNA polymerase is the enzyme responsible for synthesizing the new DNA strand. It adds new nucleotides to the 3’ end of the RNA primer.
In prokaryotes, DNA polymerase III is the main enzyme for elongation.
In eukaryotes, DNA polymerase α initiates synthesis by adding a few nucleotides, but DNA polymerase δ or ε carries out the majority of elongation.
Complementary base pairing occurs during elongation: adenine (A) pairs with thymine (T) and guanine (G) pairs with cytosine (C).
Leading vs. Lagging Strand:

Leading strand: The leading strand is synthesized continuously in the 5’ to 3’ direction towards the replication fork because it is aligned in the same direction as the helicase unwinds the DNA.
Lagging strand: The lagging strand is synthesized in the opposite direction (3’ to 5’ relative to the replication fork), so it is synthesized discontinuously in short segments called Okazaki fragments. Each Okazaki fragment requires its own RNA primer.
Elongation Process:

Leading strand: As the helicase unwinds the DNA, DNA polymerase synthesizes a continuous complementary strand in the 5’ to 3’ direction.
Lagging strand: On the lagging strand, DNA polymerase synthesizes short fragments of DNA in the 5’ to 3’ direction, which are later joined together by the enzyme DNA ligase.
3. Termination of DNA Replication
Removal of RNA Primer:

Once an Okazaki fragment is synthesized, the RNA primer must be removed so that the DNA can be joined into a continuous strand.
In prokaryotes, DNA polymerase I removes the RNA primers and fills in the gaps with DNA.
In eukaryotes, RNase H removes the RNA primers, and DNA polymerase δ fills in the gaps.
DNA Ligase:

After the RNA primers are removed, the DNA ligase enzyme seals the nicks in the sugar-phosphate backbone of the lagging strand by forming phosphodiester bonds between adjacent nucleotides. This creates a continuous strand of DNA.
Completion:

When the replication forks meet or when they reach the end of the chromosome, the replication process is considered complete.
In eukaryotes, the end of the chromosomes has specialized structures called telomeres to prevent the loss of important genetic information during replication. Telomerase extends the telomeres to maintain chromosome integrity.
Key Enzymes Involved in DNA Replication
DNA Helicase: Unwinds the DNA double helix.
Single-Strand Binding Proteins (SSBs): Prevent the single-stranded DNA from reannealing.
Topoisomerase (DNA Gyrase): Relieves strain caused by DNA unwinding.
Primase: Synthesizes short RNA primers to initiate DNA replication.
DNA Polymerase:
DNA polymerase III (prokaryotes) / DNA polymerase α, δ, ε (eukaryotes) synthesizes the new DNA strand.
DNA polymerase I (prokaryotes) removes RNA primers and fills gaps.
DNA Ligase: Joins Okazaki fragments and seals nicks in the DNA backbone.
Telomerase: Extends the telomeres of chromosomes (in eukaryotes) to prevent loss of genetic information during replication.
DNA Replication in Eukaryotes vs. Prokaryotes
Prokaryotes (e.g., E. coli):
DNA replication begins at a single origin of replication.
Replication is faster and simpler because the genome is circular, with no telomeres.
Replication is typically bidirectional.
Eukaryotes (e.g., human cells):
DNA replication occurs at multiple origins of replication along the chromosomes.
The process is slower due to the larger, more complex genome.
Replication must also deal with packaging issues, such as the DNA being wrapped around histones in chromatin.
Telomerase is required to maintain telomeres at the ends of chromosomes.
Summary of DNA Replication Process
Initiation: The DNA is unwound by helicase, and RNA primers are laid down by primase to start the synthesis process.
Elongation: DNA polymerase synthesizes new DNA strands in the 5’ to 3’ direction, using the existing DNA strands as templates. The leading strand is synthesized continuously, while the lagging strand is synthesized in Okazaki fragments.
Termination: The RNA primers are removed, and gaps are filled in by DNA polymerase. DNA ligase joins the fragments together to form complete strands.
DNA replication is a highly regulated and accurate process that ensures the faithful duplication of genetic material, which is crucial for cell division, growth, and the maintenance of genetic stability across generations.

Question 3

Describe how a base-sequence of DNA is transcribed into a base-sequence in RNA.

Answer 3

Key Steps of Transcription
Initiation:

RNA polymerase is the enzyme responsible for transcribing the DNA into RNA. The process begins when RNA polymerase binds to a specific region of the DNA called the promoter.
The promoter is a sequence of DNA located at the start of a gene. It acts as a signal for the start of transcription. In eukaryotes, additional proteins known as transcription factors assist in the binding of RNA polymerase to the promoter.
Once RNA polymerase is correctly positioned, the DNA strands are separated by the enzyme, creating a small region of single-stranded DNA known as the transcription bubble.
Elongation:

RNA polymerase moves along the template strand (also called the antisense strand) of the DNA, reading the DNA base sequence in the 3’ to 5’ direction.
As RNA polymerase moves along the template strand, it synthesizes a complementary RNA strand in the 5’ to 3’ direction (since RNA is synthesized from 5’ to 3’).
In RNA, the base uracil (U) is used instead of thymine (T), so:
A (adenine) pairs with U (uracil) in RNA.
T (thymine) pairs with A (adenine).
C (cytosine) pairs with G (guanine).
G (guanine) pairs with C (cytosine).
The RNA molecule grows as RNA polymerase adds the appropriate RNA nucleotides based on the sequence of the DNA template strand.
Termination:

Once RNA polymerase reaches a specific sequence of DNA known as the terminator, the transcription process stops. In bacteria, this is often a signal sequence in the DNA that causes RNA polymerase to detach from the DNA.
In eukaryotes, the terminator sequence is usually a region where the RNA polymerase transcribes a polyadenylation signal (poly-A signal), and the RNA molecule is cut free from the polymerase, resulting in the completion of the primary RNA transcript.
Key Players in Transcription
RNA Polymerase:

RNA polymerase is the enzyme that synthesizes RNA. It reads the DNA template strand and synthesizes the RNA strand by adding ribonucleotides.
In eukaryotes, there are different types of RNA polymerases:
RNA polymerase I: Transcribes rRNA (ribosomal RNA).
RNA polymerase II: Transcribes mRNA (messenger RNA), which encodes proteins.
RNA polymerase III: Transcribes tRNA (transfer RNA) and some small RNAs.
Promoter:

The promoter is a sequence of DNA that signals the start of transcription. It is recognized by RNA polymerase and transcription factors.
The TATA box is a common promoter sequence found in many eukaryotic genes.
Transcription Factors (in eukaryotes):

These are proteins that assist RNA polymerase in binding to the promoter. They help recruit RNA polymerase and ensure the transcription machinery is properly assembled.
Template Strand (Antisense Strand):

The template strand of the DNA is read by RNA polymerase and used as the guide for synthesizing the RNA molecule. The coding strand (sense strand) of DNA has the same sequence as the RNA, except that thymine (T) is replaced with uracil (U) in RNA.
The Process of Transcribing DNA into RNA
Let’s break it down with an example:

Example DNA Sequence:
Suppose we have the following DNA sequence in the 3’ to 5’ direction (template strand):

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3’ - ATG GCT AAC TCG - 5’
1. Binding of RNA Polymerase to the Promoter:
The RNA polymerase binds to the promoter region before the start of the gene.

2. RNA Polymerase Reads the Template Strand:
   The RNA polymerase reads the template strand of the DNA in the 3’ to 5’ direction.
   It synthesizes a complementary RNA strand in the 5’ to 3’ direction.
3. RNA Sequence:
   The RNA will be complementary to the template strand, with U (uracil) replacing T (thymine) in the RNA strand.
   Using the above DNA template strand (3’ - ATG GCT AAC TCG - 5’), the corresponding RNA sequence will be:
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   5’ - UAC CGA UUG AGC - 3’
   The RNA strand is synthesized with the bases:

A (adenine) in DNA pairs with U (uracil) in RNA.
T (thymine) in DNA pairs with A (adenine) in RNA.
C (cytosine) in DNA pairs with G (guanine) in RNA.
G (guanine) in DNA pairs with C (cytosine) in RNA.
Thus, the RNA transcript is the complementary copy of the DNA template strand but with uracil (U) replacing thymine (T).

Post-Transcriptional Modifications in Eukaryotes
In eukaryotic cells, the primary RNA transcript (also called pre-mRNA) undergoes several modifications before it becomes a mature, functional messenger RNA (mRNA) that can be translated into a protein:

5’ Capping:

A methylated guanine cap is added to the 5’ end of the RNA transcript. This cap protects the RNA from degradation and assists in ribosome recognition during translation.
Polyadenylation:

A poly-A tail (a chain of adenine nucleotides) is added to the 3’ end of the RNA. This helps stabilize the RNA and promotes its export from the nucleus to the cytoplasm.
Splicing:

Introns (non-coding regions) are removed, and exons (coding regions) are joined together to form the final mRNA. This process is carried out by the spliceosome.
After these modifications, the mRNA is ready to be transported out of the nucleus (in eukaryotes) to the ribosome for translation into a protein.

Summary of Transcription Process
Initiation: RNA polymerase binds to the promoter, and the DNA is unwound.
Elongation: RNA polymerase synthesizes an RNA strand by adding complementary RNA nucleotides to the 3’ end of the growing RNA chain.
Termination: RNA polymerase reaches the terminator, causing the RNA transcript to be released.
The final product of transcription is an RNA molecule that is complementary to the DNA template strand. This RNA can be mRNA, tRNA, or rRNA, depending on the type of gene being transcribed.

Key Differences Between RNA and DNA:
RNA uses uracil (U) instead of thymine (T) found in DNA.
RNA is usually single-stranded, while DNA is double-stranded.
The sugar in RNA is ribose, whereas in DNA, it is deoxyribose.
This process of transcription allows genetic information encoded in DNA to be converted into RNA, which can then be used to produce proteins (in the case of mRNA) or perform other cellular functions (e.g., rRNA, tRNA).

Question 4

Describe in outline the post-transcriptional processing of RNA.

Answer 4

1. 5’ Capping
   Capping involves the addition of a methylated guanine nucleotide to the 5’ end of the pre-mRNA. This modification occurs shortly after transcription begins.
   Functions of the 5’ cap:
   Protects the RNA from degradation by exonucleases.
   Helps the RNA exit the nucleus.
   Facilitates the binding of the ribosome during translation initiation.
   Enhances RNA stability.
2. Splicing
   Splicing is the process of removing introns (non-coding sequences) from the pre-mRNA and joining the exons (coding sequences) together. This results in a continuous coding sequence.
   The splicing process is carried out by a spliceosome, a complex of small nuclear RNAs (snRNAs) and proteins.
   Steps in splicing:
   The spliceosome recognizes splice sites at the junctions between introns and exons.
   It performs two cuts: one at the 5’ splice site and another at the 3’ splice site.
   The intron is excised as a lariat structure (a loop-like form).
   The exons are ligated (joined) together to form the mature mRNA.
   Alternative Splicing:
   In some cases, different exons can be included or excluded from the final mRNA, allowing for the production of multiple protein isoforms from a single gene. This is called alternative splicing and is a key mechanism in increasing protein diversity.
3. 3’ Polyadenylation (Poly-A Tail Addition)
   A poly-A tail (a string of adenine nucleotides) is added to the 3’ end of the pre-mRNA. This process is called polyadenylation and is catalyzed by the enzyme poly-A polymerase.
   Functions of the poly-A tail:
   Protects the mRNA from degradation.
   Facilitates mRNA export from the nucleus to the cytoplasm.
   Plays a role in translation initiation by assisting ribosome binding.
   Helps in the stability and longevity of the mRNA in the cytoplasm.
4. RNA Editing (Optional and Less Common)
   In some cases, the RNA sequence may undergo modifications beyond the standard splicing, capping, and polyadenylation. This is known as RNA editing.
   Examples of RNA editing include:
   Adenosine-to-inosine (A-to-I) editing: Mediated by ADAR enzymes, where adenosines are deaminated to inosines (which are interpreted as guanine during translation).
   Cytosine-to-uridine (C-to-U) editing: Occurs in some RNA molecules and can affect the RNA sequence and protein product.
   RNA editing can alter the coding sequence of the mRNA, potentially leading to the production of different protein isoforms.
5. mRNA Transport
   After processing, the mature mRNA is transported from the nucleus to the cytoplasm for translation. This is a regulated process that involves interactions between mRNA and various proteins.
   The nuclear pore complexes recognize and transport mRNA that has been properly processed (capped, spliced, and polyadenylated).
6. RNA Surveillance and Quality Control
   Cells have mechanisms to ensure that only properly processed RNA is exported and translated. If mRNA is improperly spliced or contains mutations, it can be recognized and degraded by nonsense-mediated decay (NMD) and other quality control mechanisms.
   Exon-junction complexes are deposited at exon-exon boundaries during splicing, and these can signal to the cell if there are mistakes in the splicing process, leading to mRNA degradation before it reaches the cytoplasm.
   Summary of Post-Transcriptional Modifications
   5’ Capping: Addition of a methylated guanine cap to the 5’ end of the mRNA.
   Splicing: Removal of introns and joining of exons to form a continuous coding sequence.
   Polyadenylation: Addition of a poly-A tail to the 3’ end of the mRNA.
   RNA Editing: Optional modifications to the RNA sequence, such as base changes.
   mRNA Transport: Processed mRNA is exported from the nucleus to the cytoplasm.
   RNA Surveillance: Quality control mechanisms ensure only properly processed RNA is translated.

Question 5

Describe the translation of mRNA.

Answer 5

Translation can be broken down into several key steps: Initiation, Elongation, and Termination. Let’s go through the process in detail.

1. Initiation of Translation
   Goal: To assemble the translation machinery (ribosome, mRNA, and tRNA) and start protein synthesis at the correct codon.

Key Players:

mRNA: The messenger RNA that carries the genetic code from the DNA to the ribosome.
Ribosome: A complex of ribosomal RNA (rRNA) and proteins that provides the site for protein synthesis.
In eukaryotes: The ribosome is made up of a large subunit (60S) and a small subunit (40S).
In prokaryotes: The ribosome is made up of a large subunit (50S) and a small subunit (30S).
Transfer RNA (tRNA): Small RNA molecules that carry amino acids and have anticodons that are complementary to the mRNA codons.
Initiator tRNA: A special tRNA that carries methionine (in eukaryotes) or formylmethionine (in prokaryotes) and binds to the start codon of the mRNA.
Steps in Initiation:

mRNA Binding: The small ribosomal subunit binds to the mRNA near the 5’ cap in eukaryotes (or the Shine-Dalgarno sequence in prokaryotes).
Formation of the Initiation Complex:
The initiation factors help the small subunit recognize the mRNA and bind to it.
The initiator tRNA (charged with methionine in eukaryotes or formylmethionine in prokaryotes) pairs with the start codon (usually AUG) on the mRNA. The anticodon of the initiator tRNA pairs with the codon on the mRNA.
Ribosome Assembly: The large ribosomal subunit (60S in eukaryotes or 50S in prokaryotes) binds to the small subunit, forming the complete ribosome.
Start Codon Recognition: The initiator tRNA, carrying the first amino acid (methionine), is now positioned in the P site of the ribosome.
At the end of initiation, the ribosome is ready to begin elongating the polypeptide chain.

2. Elongation of the Polypeptide Chain
   Goal: To add amino acids, one at a time, to the growing polypeptide chain.

Key Players:

tRNA: Each tRNA carries a specific amino acid and has an anticodon that is complementary to the mRNA codon.
Aminoacyl-tRNA: A tRNA molecule that is charged (bound) to its respective amino acid.
Elongation factors: Proteins that assist in the elongation process by facilitating tRNA binding, peptide bond formation, and ribosome movement along the mRNA.
Steps in Elongation:

Codon Recognition:

The ribosome moves along the mRNA, reading each codon (a sequence of three nucleotides) in the 5’ to 3’ direction.
The A site of the ribosome (the aminoacyl site) receives a new aminoacyl-tRNA that matches the mRNA codon with its anticodon. Each tRNA carries a specific amino acid.
Peptide Bond Formation:

The ribosome catalyzes the formation of a peptide bond between the amino acid at the P site (the peptidyl site) and the amino acid at the A site.
This reaction is facilitated by the ribosomal RNA (rRNA) in the large subunit of the ribosome, which acts as a ribozyme.
The amino acid in the P site is transferred to the growing polypeptide chain in the A site.
Translocation:

After the peptide bond is formed, the ribosome moves along the mRNA in a process called translocation.
The ribosome shifts by one codon to the 3’ direction along the mRNA. The tRNA in the P site moves to the E site (exit site), where it is released.
The tRNA in the A site moves to the P site, making room for a new tRNA in the A site.
This cycle repeats, adding one amino acid at a time to the growing polypeptide chain.
3. Termination of Translation
Goal: To end the translation process when a stop codon is encountered and release the newly synthesized protein.

Key Players:

Stop codons: The mRNA contains three stop codons (UAA, UAG, and UGA), which do not code for any amino acid.
Release factors: Special proteins that recognize the stop codons and catalyze the release of the polypeptide from the ribosome.
Steps in Termination:

Stop Codon Recognition: When the ribosome reaches one of the stop codons on the mRNA, there is no tRNA that corresponds to the stop codon. Instead, a release factor (such as RF1, RF2 in prokaryotes or eRF in eukaryotes) binds to the stop codon in the A site of the ribosome.

Polypeptide Release:

The release factor catalyzes the hydrolysis of the bond between the polypeptide and the tRNA in the P site, releasing the newly synthesized protein.
The ribosome dissociates into its large and small subunits, and the mRNA is released.
4. Post-Translational Modifications (Optional)
Once translation is complete, the newly synthesized polypeptide chain may undergo various post-translational modifications to become a functional protein. These modifications can include:

Phosphorylation: Addition of phosphate groups.
Glycosylation: Addition of sugar molecules.
Acetylation: Addition of acetyl groups.
Proteolytic cleavage: Cutting the polypeptide chain into smaller functional units.
Folding: The polypeptide folds into its three-dimensional structure with the help of chaperone proteins.
Summary of Translation Steps
Initiation: The ribosome assembles on the mRNA, the initiator tRNA binds to the start codon (AUG), and the large ribosomal subunit joins.
Elongation: tRNAs bring amino acids to the ribosome, where the codon-anticodon pairing ensures correct amino acid addition. Peptide bonds are formed, and the ribosome moves along the mRNA.
Termination: When a stop codon is encountered, release factors cause the ribosome to release the newly synthesized protein and disassemble.
Final Product: A newly synthesized polypeptide chain, which will fold into a functional protein after any necessary post-translational modifications.

Key Points
mRNA provides the template for translation.
tRNA brings amino acids and matches codons with their anticodons.
Ribosomes are the sites of protein synthesis, facilitating the decoding of mRNA and assembly of the polypeptide.
Translation involves three main stages: initiation, elongation, and termination.
Post-translational modifications are often required for a protein to become fully functional.