CENTRAL DOGMA OF MOLECULAR BIOLOGY

Central Dogma

It is the flow of genetic information in the cell.

The mechanism on how the information is transferred to a cell is based on this.

Proofreading and Repair

During replication of DNA, what other processes are taking place?

Hint: it ensures that there are no incorrect base pairs in the sequence

• Transcription

• Nucleus

DNA undergoes ____ to synthesize RNA. This occurs in the ____.

Translation

RNA will go out of the nucleus to the cytoplasm and will undergo ____ to form proteins.

False. Both Prokaryotes and Eukaryotes contain genetic information.

True or False.

Only eukaryotes contain genetic information.

1. Prokaryotes

2. Eukaryotes

Determine if it is Prokaryotes or Eukaryotes.

1. It has a circular double-stranded DNA.

2. It has a linear double-stranded DNA.

DNA replication

It is a semiconservative process wherein the parent cell will separate into daughter cells.

There is a separation of the strands of the double helix, and synthesis of two daughter strands complementary to the two parental templates.

1. Unwinding and Separation

2. Unwound; Nucleases

3. 5’ to 3’ (Forward) and 3’ to 5’ (Reverse)

4. Errors

The challenges in duplication of circular double-stranded DNA.

1. There is a continuous ____ and ____ of the 2 DNA strands.

2. There is a protection of ______ portions from attack by _____ that attack single-stranded DNA or nucleic acids.

3. Synthesis of the DNA template from one ____ strand and one ____ strand.

4. Efficient protection from ____ of replication.

Origin of Replication or Origin

The site wherein the DNA double helix will unwind.

This is where polynucleotide chains are formed.

Bidirectional

The polynucleotide chains are synthesized in both directions of the origin (both sides). This indicates that DNA replication is ___ in most organisms.

Replication Forks

This is where the 2 strands of DNA separate.

2

How many replication forks are present in each origin of replication?

True

» Eukaryotes can have several origins of replication because of its complex structure. Some DNAs have 3 origins, with 6 replication forks (since each origin have 2 forks).

True or False.

Eukaryotes can have several origins of replication because of its linear DNA.

1. Leading strand

2. Lagging strand

1. What strand is 5’ to 3’?

2. What strand is 3’to 5’?

Leading strand

This strand is synthesized continuously in the 5’ to 3’ direction towards the replication fork.

• Lagging

• Semidiscontinuously

• Okazaki fragments

The ____ strand is synthesized ______ through ___ _____ in the 5’ to 3’ direction, but away from the replication fork.

DNA ligase

The lagging strand (Okazaki) fragments are joined by the enzyme _____

DNA-directed DNA polymerase

It is responsible for the synthesis of new strands of DNA from the template.

RNA primer

DNA polymerase requires a _____ to provide a 3’ Hydroxyl (-OH) terminus in which to add new nucleotides.

Primase

In the formation of the RNA primer, the enzyme involved is ____, which will copy a portion of the template and contains a 3’ Hydroxyl end.

Complementary

The lagging strand is ____ to the template.

1. Deoxynucleotide Triphosphates (DNTPs)

   » dATP, dTTP, dGTP, dCTP

2. Magnesium (Mg2+)

3. RNA primer

What are the requirements for DNA polymerase function?

RNA primer

A short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication.

1. DNA Polymerase III

2. DNA Polymerase I

3. DNA Polymerase II, IV, and V

DNA POLYMERASE OF E.COLI

1. Responsible for the polymerization of the DNA strand

2. Responsible for the repair and patching of the DNA.

3. Responsible for proofreading and repair of the enzymes.

DNA Polymerase I

The only exonuclease that has a 5’ to 3’ direction in E.coli

1. DNA gyrase (topoisomerase II)

2. ATP hydrolysis

1. Relieves the tension by changing the DNA into negatively supercoiled DNA

2. What process does this use?

Positively supercoiled

If there is tension, the DNA becomes _____ ____, hence it has to unwind. Else, it will be hard to replicated DNA.

1. Primosome

2. Create RNA primers on the single-stranded DNA during replication.

1. A protein complex of primase, helicase, and single-stranded DNA binding proteins. It moves along the lagging strand.

2. What is its function?

Primase

A RNA polymerase that catalyzes the copying of a short stretch of DNA template strand to produce RNA primer sequence.

DNA helicase

It separates the double helix (strands) of the DNA by removing the hydrogen bonds.

Single-stranded DNA Binding Proteins (SSB proteins)

It stabilizes the single-stranded DNAs to not turn into an allele and to protect it from nucleases.

Replisome

1. A replication complex. It is a large protein complex that carries out the DNA replication. It starts at the origin.

Other information: it’s also responsible to ensure that the movement of DNA polymerase is in coordination.

DNA Polymerase III

Helicase

SSBs (Single-stranded DNA Binding proteins)

Primase

What is replisome composed of?

1. DNA Polymerase III

2. 3’-OH of RNA Primer

1. Synthesis and linking of new DNA strands (or DNA replication) will be begun by?

2. The newly formed DNA will be linked to?

1. Replicate DNA

2. Alpha, Beta, Delta, Epsilon, Gamma

EUKARYOTES GENERAL INFORMATION

1. Before cell division, eukaryotes must?

2. What are their DNA polymerases?

1. Gamma – Mitochondria

2. Alpha

3. Epsilon and Beta

EUKARYOTIC DNA POLYMERASES 1

1. Which of them is not found in the nucleus? Where is it found?

2. Which DNA Polymerase has Primase?

3. What DNA Polymerase is involved in repair?

1. Beta

2. Alpha, Beta

3. Beta

4. Alpha, Beta

EUKARYOTIC DNA POLYMERASE 2

1. What DNA Polymerase is not involved in replication?

2. What DNA Polymerase do not have 3’ 5’ exonuclease?

3. Low fidelity

4. Low processivity

1. Alpha

2. Beta

3. Gamma

4. Delta

5. Epsilon

EUKARYOTIC DNA POLYMERASES 3

1. A polymerizing enzyme

2. A repair enzyme

3. Involved in Mitochondrial DNA synthesis

4. Main polymerizing enzyme

5. The leading strand replication enzyme

1. DNA Polymerase II

2. DNA Polymerase I

3. DNA Polymerase III

PROKARYOTIC DNA POLYMERASES

1. It is a repair enzyme.

2. It is involved in synthesis, proofreading, repair, and removal of RNA primers.

3. The main polymerizing enzyme.

1. Eukaryotic (Prokaryotes polymerases are also exonucleases).

2. Prokaryotic (Eukaryotes have several origins)

3. Prokaryotic (Eukaryotes have 150-200 residues long)

4. Eukaryotic (Prokaryotes do not have proteins complexed to DNA)

Determine if it is Prokaryotic or Eukaryotic.

1. Polymerases are not exonucleases.

2. One origin of replication

3. Okazaki fragments have 1000-2000 residues long.

4. Histone complexed to DNA.

Tus proteins

The recognition site for a sequence-specific DNA binding protein.

Orientation of the termination sequence

Tus protein orientation relies on?

• One direction

• Opposite direction

Tus proteins allow a replication fork to pass if the fork is moving in ____ _____, but blocks progress if the fork is moving in ____ ____.

1. Transcription and Translation

2. Accessing the genome

3. Protein degradation

1. Gene expression includes?

2. What does it begin with?

3. It ends in?

1. Transcription

2. DNA-Dependent RNA Polymerase

1. This process uses the DNA template to form RNA.

2. What is its primary enzyme?

1. Transcription - DNA-dependent RNA polymerase

   Replication - DNA-directed DNA polymerase

2. Replication. Transcription uses NTPs.

3. Replication. It uses both 5’ to 3’ and 3’ to 5’. Transcription uses only the lagging strand.

4. Transcription

TRANSCRIPTION vs REPLICATION

1. What are their corresponding enzymes?

2. Which one of them uses dNTPs as substrate? What does the other use?

3. Which one uses 5’ to 3’ replication?

4. Which one does not require a primer?

1. DNA nontemplate (coding) strand

2. DNA template (antisense) strand

1. What DNA template is 5’ to 3’ (CGCTATAGCGTTT)?

2. What DNA template is 3’ to 5’ (GCGATATCGCAAA)?

1. Template (antisense) strand - Reverse

2. Nontemplate (coding) strand - Leading

1. The strand that serves as the template for RNA synthesis.

2. Identical to the RNA transcribed from the gene, with U instead of T in DNA.

Bacterial DNA-dependent RNA polymerase

• Alpha 1, Alpha 2, Beta, Beta prime, Omega (β′, β, α^Ι, α^II, and ω) - core subunits

• Sigma factor (σ) - sixth subunit

It is a multi-subunit enzyme that is made up of five core subunits and a sixth subunit, namely?

1. Beta prime

2. Sigma factor

Bacterial DNA-Dependent RNA Polymerase

1. It is thought to be a catalytic subunit.

2. It directs the core complex to specific binding sites on the DNA.

1. RNA Polymerase I, II, III

2. 1 - Nucleolus

   2 and 3 - Nucleoplasm

1. What are the 3 distinct nuclear DNA-dependent RNA polymerases in Mammalian cells?

2. What are their corresponding compartment?

RNA Polymerase II

MATCHING TYPE.

What Mammalian DNA-Dependent RNA polymerase transcribes the following genes?

• Protein-coding genes, most small nuclear RNA (snRNA) genes

RNA Polymerase I

MATCHING TYPE.

What Mammalian DNA-Dependent RNA polymerase transcribes the following genes?

• 28S, 5.8S, and 18S ribosomal RNA (rRNA) genes

RNA Polymerase III

MATCHING TYPE.

What Mammalian DNA-Dependent RNA polymerase transcribes the following genes?

• Genes for transfer RNA (tRNAs), 5S rRNA, U6-snRNA, small nucleolar RNAs, small cytoplasmic RNAs

12

RNA Polymerase II consists of how many subunits?

Initiation

Elongation

Termination

What are the three phases of Transcription?

Promoter

A DNA sequence to which polymerase binds prior to initiation of transcription.

• Pribnow Box (-10 region)

• -35 region

• UP element

Promoter regions in Prokaryotes consist of?

Binding

Step 1 of the Transcription: Initiation Phase

Transcription bubble (17 bp long)

RNA polymerase unwinds the double helix near the -10 consensus sequence to form the?

Initiation - Transcription

RNA polymerase catalyzes the formation of a phosphodiester bond between 2 initial ribonucleoside triphosphates

More than 10 nucleotides

σ (Sigma) factor dissociates after addition of?

Promoter clearance

RNA polymerase undergoes structural changes to its elongation conformation followed by movement of the transcription complex away from the promoter

• Upstream elements (enhancers and silencers)

• Core promoter

• Downstream regulators

Promoters include?

• Activators

• Repressors

Who directs transcription regulation during initiation?

• Activators

• cAMP Receptor Protein (CRP)

• They facilitate either RNA polymerase binding or steps further along in the initiation process

• Give example

• Repressors

• Lac repressor

• They block polymerase activity

• Give example

Transcription Activators

IN EUKARYOTES DNA PACKAGING

they bind to enhancer sequences to help attract RNA polymerase II to the transcription start site

Mediators

IN EUKARYOTES DNA PACKAGING

It allows the activator proteins to communicate properly with RNA polymerase II and General Transcription Factors

Chromatin modifying enzymes

IN EUKARYOTES DNA PACKAGING

They allow greater accessibility to the DNA allowing assembly of transcription initiation complex

1. Closed promoter complex

2. Open promoter complex

What promoter complex?

1. Recognition of promoter region by sigma factor

2. Unwinding of the DNA

-10 box

The opening of the double helix starts at?

Elongation

RNA polymerase directs the sequential addition of ribonucleotides to the growing RNA chain in the 5’ to 3’ direction

• Elongation

• Purine ribonucleotide triphosphate (PRT)

• What phase?

• Addition of ___ ____ ____ as the first base of RNA transcript.

50 - 90 nt/s

The rate of RNA polymerase

False. They do not have this activity.

True or False. RNA Polymerase have 3’-5’ exonuclease activity.

Water molecule replaces pyrophosphate

A nucleoside monophosphate is then released

If an incorrect ribonucleotide is added, the polymerase can perform an excision reaction that resembles the reverse of the polymerization reaction through?

DNA gyrase

In bacteria, this remove the superhelical tension generated by DNA unwinding during elongation.

ρ-Dependent Mechanism (Extrinsic Termination)

This termination weakens the interaction between the template and the transcript.

P-Factor

It recognizes the termination region.

ρ-Independent Mechanism (Intrinsic Termination)

This termination site contains palindromes in the template strand; the newly synthesized RNA forms a hairpin loop

GC-rich inverted repeat

It allows a hairpin to form at ~15 to 20 nt before the end of the transcript and destabilizes the RNA-DNA hybrid

String of As in template strand

This causes transcription to pause

• Nucleoid

• Cytosol

Prokaryotic mRNAs are synthesized on the bacterial ___ in direct contact with the ___ and are immediately available for translation.

mRNA maturation

• 5’

• Introns

• Polyadenylation

Eukaryotic mRNA synthesis is also called?

It includes addition of (__) capping, splicing of (__), and (__) at 3’ tail

1. To protect the mRNA from exonucleases

2. Add GTP by guanylyl transferase » Methylation of G by Guanine methyltransferase

1. Why do we need to cap 5’ of mRNA?

2. How to cap 5’ of mRNa?

1. GU-AG motifs

2. 5’ splice site

3. 3’ splice site

1. Introns are made up of?

2. What site is 5’-AG↓GUAAGU-3’?

3. What site is 5’-PyPyPyPyPyPyNCAG↓-3’?

1. 5’

2. 3’ ; exons

STEPS IN SPLICING PATHWAY

1. Cleavage of the _____ site

2. Cleavage of the _____ site and joining of the ___

Spliceosome

This is a complex that is made up of snRNAs with snRNPs.

Translation

The synthesis of polypeptides using the genetic code from RNA.

Genetic code

It specifies how a mRNA sequence is translated to a polypeptide.

1. Triplet

2. Nonoverlapping

3. Commaless

4. Degenerate

5. NOT universal

5 Major Features of the Genetic Code

• RNAs (mRNA, tRNA, rRNA)

• Ribosome

• Protein factors (IFs, EFs, RFs)

• Activated substances

Requirements for Translation

1. tRNA

2. [ADVAT]: Acceptor arm, D arm, V loop, Anticodon loop, TΨC arm

3. Three leaf clover

1. It serves as adaptor molecule that provides physical and informational link between mRNA and the polypeptide being synthesized.

2. What is its structure composed of?

3. What does it resemble?

Aminoacylation

This is the charging of tRNA

1. 3’

2. 5’

1. 16s Ribosomal RNA will be at what end of the strand?

2. mRNA 30S ribosomal subunit will be at what end of the strand?

1. Amino acid group

2. Aminoacyl, Synthetase enzyme

1. To prepare a mRNA for translation, ____ must be added to tRNA.

2. To activate tRNA, there is need for tRNA, ___, and ____ enzyme.

C-C-A at atheir 3’ prime ends

All tRNAs must contain the same bases, specifically?

Adenosine

What is the target for the aminoacylation (charging)?

Wobble base pairing

A non-standard base pairing between the base at 5’ end of anticodon and the base at 3’ end of codon.

It does not follow the usual Watson-Creek base pair rules.

It allows flexibility.