Central Dogma of life is a theory that describes how the genetic information stored in the nucleus in the form of DNA, is used to synthesize mRNA and then proteins.
DNA is double-stranded while mRNA is single-stranded in most cases. DNA strands are anti-parallel such that one strand runs from 5′ end to the 3′-end , while the complementary strand runs from 3′ end to the 5′ end.
For DNA to form RNA and then protein, the two strands must be unzipped. The unwinding of the two-strand is carried out by an enzyme called helicase. Primase adds RNA primers on the 5′ end of each strand.
Steps in central dogma
DNA in the nucleus – mRNA that is transported out of the nucleus to the cytoplasm – the last step is protein synthesis using mRNA within the ribosomes
Transcription
The process begins within the nucleus, using RNA polymerase. On the gene, promoter sequence guides RNA polymerase where to begin the transcription process.
RNA polymerase is the main enzyme that is responsible for adding RNA nucleotides to the growing strand. Transcription does not use RNA primer as the case with the replication of DNA.
Only one strand of DNA is used to synthesize mRNA and this strand is usually called the template strand. The process proceeds from the 5′ end of the template strand, forming a strand that proceeds in 3′-5′ direction.
Polymerases and other enzymes, transcription factors and consensus sequences transcribe the template strand of DNA to form mRNA
The formed mRNA is usually immature and needs processing. Examples of this processing include the removal of non-coding regions called introns.
The coding regions (exons) are joined together to form the mature mRNA that has to undergo post-transcription modifications as we will look at in the next lesson.
Pre-mRNA to mature messenger RNA
- When the mRNA is mature and all post-transcriptional modifications have occurred, it is transported out of the nucleus and into the cytoplasm
- It is moving to the cytoplasm because ribosomes which are sites for protein synthesis are located in the cytosol
Translation
The translation process occurs in the ribosome and forms amino acids from the mRNA sequence.
The process requires transfer RNA which carries amino acid anticodons. tRNA transfers amino acids on the growing strand. An amino acid is added on every three nucleotides that make a codon.
After amino acids are formed, the peptide bond joins the amino group with the carboxyl group of each amino acid to form a polypeptide.
Post-translational modification
- Formation of disulfide bonds – They are used to stabilize the tertiary and the quaternary structure of proteins.
- Proper folding – folding proper confirmation in the lumen of ER
- Addition biochemical groups
- Specific proteolytic cleavages – This is done by peptidases, proteases enzymes which break peptide bonds to remove N-terminal methionine residue
- Assembly into multimeric proteins.
Revision
Gene Expression
The process of converting archived information into molecules that actually do things.
One Gene, One Enzyme Hypothesis
Each gene encodes a singe enzyme. Today, we know the idea is generally (but not always) correct.
Metabolic Pathway
A series of steps found in biochemical reactions that help convert molecules or substrates (such as sugars) into different, more readily usable molecules.
Genetic Screen
Any technique for picking particular types of mutants out of many randomly generated mutants.
Messenger RNA (mRNA)
Carry information out of the nucleus from DNA to the site of protein synthesis. One of the many distinct types of RNA.
RNA Polymerase
Polymerizes ribonucleotides into strands of RNA. Like DNA Polymerase, RNA Polymerase uses a DNA strand as a template to specify which complementary nucleotide to add to the growing newly synthesized strand
Central Dogma
The flow of information in cells.
DNA –Transcription–> mRNA –Translation–> Proteins
Transcription
The process of using a DNA template to make an RNA molecule that has a base sequence complementary to the DNA. DNA is transcribed to RNA by RNA Polymerase.
Translation
The process of using the information in mRNA to synthesize proteins. Information stored in the messenger RNA is translated into proteins by ribosomes.
Reverse Transcriptase
A viral enzyme that synthesizes a DNA version of the RNA genes. (In these viruses, information flows from RNA to DNA)
Hydrolyze
To break down (Use water to break bonds)
Genetic Code
The rules that specify the relationship between a sequence of nucleotides in DNA or RNA and the sequence of amino acids in a protein.
Codon
A group of 3 bases that specifies a particular amino acid
Start Codon
Signals that protein synthesis should begin at that point on the mRNA molecule
Methionine (Met) – mRNA code is AUG
Stop Codon
Also known as termination codons.
Don’t code for any amino acid, but signal the end of the translation when the protein is complete
mRNAs – UAA, UAG, UGA
What is supercoiling?
When DNA coils back on itself (coils within coils)
Why is supercoiling important?
It condenses DNA to fit more in a small space (i.e. the cell)
Is transcription with translation useful in any way?
Yes, mRNAs would be wasted, but transcription also gives us tRNA, rRNA, gRNA which have other uses.
Is translation without transcription useful in any way?
No, because you can’t translate without mRNA
Template Strand
The strand of DNA that’s read by the enzyme (RNA polymerase)
Coding (Non-Template) Strand
With one exception (A-U), its sequence matches the sequence of the RNA that’s transcribed from the template strand and codes for a polypeptide.
3 Phases of Transcription
- Initiation
- Elongation
- Termination
Sigma
A protein that must bind to the polymerase before transcription can begin
Holoenzyme
Together, bacterial (prokaryotic) RNA polymerase and sigma form this “whole enzyme”
Core Enzyme
Contains active site for catalysis, and other required proteins (i.e. sigma)
Promoters
Regions of DNA that promote the start of transcription
Downstream
DNA that’s located in the direction RNA polymerases move during transcription
Upstream
DNA located in opposite direction RNA polymerases move during transcription
Elongation
The enzyme catalyzes the addition of nucleotides to the 3′ end of the growing RNA at the rate of about 50 nucleotides per second
Termination
Ends Transcription
What term is used to describe the process of converting stored information into molecules that actually do things in the cell?
Gene Expression
In figure 16.1: If you mutated the cell such that there is no Enzyme 2 and provided the cell with citrulline…would the cell survive?
Yes
In figure 16.1: If you mutated the cell such that there is no Enzyme 3 and provided the cell with ornithine…would the cell survive?
No
Are all RNA molecules mRNA?
No, there are multiple other kinds of RNA molecules in most cells
What amino acid is encoded by the codon UAU?
Tyrosine
What amino acid is encoded by the codon GAC?
Aspartic Acid
What amino acid is encoded by either the codon AGG or the codon CGC?
Arginine
In eukaryotic cells, transcription mostly occurs
In the nucleus
RNA polymerase is an enzyme that
Creates phosphodiester bonds between ribonucleic acid monomers
A promoter is
A part of a nucleic acid polymer
The promoter is a region of DNA. In most prokaryotic organisms
Has two binding sites on it that can be bound by sigma protein
To do its job well, RNA polymerase must
Break H-bonds
In the case of transcription, the word ‘template’ means
The DNA strand that is used as a guide to create a new RNA strand
If the template strand of DNA has a Thymine nucleotide, the new RNA will have a(n)
Adenine
Which general statement is typically correct about molecular processes in biology?
Eukaryotic processes are similar to prokaryotic processes but are often more complicated
If a promoter is extremely strong, then
Sigma will bind to it often
5 Main Properties of the Genetic Code
- Redundant
- Unambiguous
- Non-overlapping
- Nearly universal
- Conservative
Why is DNA more likely to be the information storage molecule than RNA?
The double-stranded DNA allows for replication of information, whereas single-stranded RNA has the potential of being lost forever.
Why are proteins more likely the work molecule versus RNA?
Proteins are more versatile, because they can be polar/nonpolar, and they have distinct R-groups
Why does the new mRNA always construct in the 5′ to 3′ direction?
Only the 3′ side is reactive, because it has hydroxyl groups with negative charges.
What changes would disrupt the binding of sigma to the promoter?
Changing the -10 site
Changing the primary sequence of sigma
Removing all upstream from -5
Rudder
In the RNA Polymerase, responsible for opening the DNA by breaking the H-bonds between the nucleotides.
Zipper
In the RNA Polymerase, responsible for breaking the DNA-RNA bonds, and allows for the RNA to leave.
Ribosomes
Site of protein synthesis
Polyribosomes
When 2 or more ribosomes simultaneously translate one mRNA
Transfer RNA (tRNA)
Serve as chemical go-betweens that allow amino acids to interact with an mRNA template.
Anticodon
A triplet of ribonucleotides able to form base pairs with the codon for the amino acid in mRNA
Aminoacyl tRNA
When a tRNA is linked to its amino acid
How do amino acids become linked to tRNAs?
There needs to be adapter molecules; 1 per every codon
What are the important parts of the tRNA?
3 base pairs that bond to mRNA (the anticodon)
The binding site for the amino acid
How is tRNA charged?
It’s ready to do work; it has two charges (zwitterion)
How do enzymes create charged tRNA?
Use of ATP binding?
Disadvantage(s) of Eukaryotes in terms of Transcription/Translation occurrence with a nucleus
There’s no coupling occurring; slow
Advantage(s) of Eukaryotes in terms of Transcription/Translation occurrence with a nucleus
Protection for the DNA
Coupling
Transcription and translation occurring simultaneously
What’s the problem with a 2-base code?
There wouldn’t be enough combinations for how many amino acids there are.
What’s the problem with a 4-base code?
It would be redundant/wasteful because there would be too many combinations, that it would be a waste of energy to produce so many.
Information necessary to translate DNA
Start/stop codons
Directionality (promoter; -10 and -35)
Strand Directionality (5′-3′ direction)
Sequence
Mutation
Any permanent change in an organism’s DNA
Point Mutation
A mutation that alters the sequence of one or a small number of base pairs.
Silent Mutation
A point mutation that doesn’t change the amino acid sequence of the gene product, so there’s no change in the phenotype. Neutral with respect to fitness.
Frameshift Mutation
The addition or deletion of a nucleotide which results in the reading frame shifting, altering the meaning of all subsequent codons; almost always deleterious.
Nonsense Mutation
Change in nucleotide sequence that results in an early stop codon. It leads to mRNA breakdown or a shortened polypeptide.
Missense Mutation
A change in nucleotide sequence that changes the amino acid specified by the codon. It changes the primary structure of protein; but it can be beneficial, neutral or deleterious.
Which is most likely to impact fitness, and why?
-Change in DNA of a skin cell
-Change in DNA of a sperm cell
-Change in a protein in a sperm cell
-Change in an mRNA in a skill cell
All are likely to impact fitness, however, the changing in DNA of a sperm cell is most likely to impact fitness because it is the most heritable, and the DNA is the source of all information. So if the DNA gets mutated, all future proteins and DNAs synthesized by that DNA will be affected.
Free Radical
Increased energy of an electron, increases possible collisions – if collide with DNA can cause mutations.
A UV ray causes a mutation that codes for a substitution in a single amino acid of an enzyme. What’s the most likely outcome in terms of enzyme function?
The enzyme function will most likely decrease, because it could potentially change the shape of the polypeptide, possibly blocking its binding site, and rendering the protein useless.
What’s the most likely outcome in terms of enzyme function of a deletion of 1 amino acid?
- It could make a SMALL impact, because it is just 1 amino acid, and could not be at a location of the polypeptide binding site.
- It could also be a LARGE impact, because it could affect how the polypeptide binds correctly in the active site, and could also be very important to structure.
Why does a change in an R-group have the biggest impact?
The change in the R-group could change the charge in a nonpolar protein core, which could affect the overall structure of the protein.
It could also change the shape (proline ring shape vs. leucine strand)
Origin
Starting point of replication of new strands.
Why eukaryotic chromosomes have multiple origins?
It’s more efficient –> faster
Important for bigger chromosomes
Why does replication go in both directions?
Both have a 5′ to 3′ orientation
Topoisomerase
Breaks and rejoins the DNA double helix to relieve twisting forces caused by the opening of the helix.
Helicase
Breaks the H-bonds between base pairs in order to open the double helix of the DNA
Primase
Adds small RNA primers
Why can’t DNA replication start without a primer?
Enzyme needs a 3′ OH (limitation of the actual enzyme)
Single-Stranded DNA-binding Proteins
SSBP
Protects single stranded DNA from breaks
(Also stops DNA from snapping shut)
What would be a phenotype of a mutation in topoisomerase?
Tight DNA, could break or prevent replication
What would be a phenotype of a mutation in helicase?
No opening of the DNA, no replications
Sliding Clamp
Keeps the DNA Polymerase on the DNA
3 Enzymes Responsible for Opening the Helix in DNA Replication
- Helicase
- Single-Stranded DNA-binding Proteins
- Topoisomerase
3 Enzymes Responsible for Leading Strand Synthesis
- Primase
- DNA Polymerase III
- Sliding Clamp
5 Enzymes Responsible for Lagging Strand Synthesis
- Primase
- DNA Polymerase III
- Sliding Clamp
- DNA Polymerase I
- Ligase
DNA Polymerase III
Extends the leading strand, also extends the Okazaki fragments
DNA Polymerase I
Removes RNA primer and replaces it with DNA
DNA Ligase
Catalyzes the joining of the Okazaki fragments into a continuous strand by patching phosphodiester bonds.
What would be a phenotype of a mutation in DNA ligase?
Still some breaks in DNA, and the DNA would be vulnerable to new DS breaks
Replication Fork
Y-shaped region where the parental DNA double helix is separated into single strands and copied.
