DNA and RNA: Structure, Function, and Replication - Biochemistry of the Genome, Study notes of Microbiology

Download DNA and RNA: Structure, Function, and Replication - Biochemistry of the Genome and more Study notes Microbiology in PDF only on Docsity!

Biochemistry of the Genome 6/

Nucleic Acids like DNA/RNA are the 4th major macromolecules found in living organisms DNA (deoxyribose nucleic acid)

• Responsible for storing all the hereditary information in the cell
• The genetic information that is stored in the form of DNA can be copied during DNA replication and then transmitted from mother to daughter cells during cell division
• Building blocks are monomers of nucleic acids - nucleotide
  • Ex: deoxyribose nucleotides
  • All consists of:
    • A nitrogenous base
      • 6 carbon structure: pyrimidines
        • Cytosine, thymine
      • 5 carbon ring that is fused to a 6 carbon ring structure: purines
        • Adenine, guanine
    • 5 carbon sugar (ex: deoxyribose sugar)
    • Phosphate group (chain of either 1-3 molecules) Naming
• Ex: picture on the side
• The unit that is comprised of the nitrogenous base and the deoxyribose sugar: nucleoside
• Since in the picture, the sugar is with an adenine base, the name of the molecule is deoxyadenosine
• Nucleoside is then bound to one or more phosphate groups
• Since it is bound to one phosphate group in this picture, the name is:
• Deoxyadenosine monophosphate
• If bound to two phosphate groups, the name would be:
• Deoxyadenosine diphosphate Individual nucleotides triphosphates will combine or polymerize to make up a chain of nucleotides which make up the nucleic acid molecule
• Individual nucleotides, when joined together, 2 phosphates will be lost during the polymerization process
• Only 1 phosphate will be left
• Covalent bond is going to be formed with adjective nucleotides known as 5’-3’ phosphodiester bond

• Even though RNA is single stranded, still can pair the nitrogenous bases
  • Pairing rules:
    • Guanine and cytosine
    • Adenine and uracil
  • Results in the RNA molecules folding into predictable 3D molecules
    • Leads to different functions
• Functional differences
• DNA molecule is stabilized by the base pairings that occurs between the 2 strands
• Stable molecule for the long-term storage of genetic information
• RNA molecule is less stable than the DNA molecule
• Suited for short-term functions Function of RNA molecules
• Cells access the information stored in the DNA by creating RNA molecules that direct the synthesis of proteins
• 3 types of RNA molecules
• Messenger RNA (mRNA)
• RNA copy of a particular DNA sequence that encodes the instructions of creating a particular protein
• mRNA produced in a process called transcription
• Attaches to the ribosome
• Ribosomal RNA (rRNA)
• Carries out the synthesis of proteins based on the instructions provided by the mRNA molecule through the process called translation
• Transfer RNA (tRNA)
• Attached to a single amino acid
• Role in translation is to bring the particular amino acid to the ribosome and add it to the growing polypeptide chain that will fold into a protein that is being built at the ribosome RNA as hereditary information
• Not used for the long term storage of the genetic information
• Many viruses have RNA genome s instead of DNA genomes
• RNA is involved in the genetic information for these viruses
• They can consist of single strand or double strand RNA molecules
• Double stranded RNA - exclusively seen in viruses and not seen in any living organisms
• Ex: SARS-COV 2
• Have a single stranded genome Genomes
• Collection of all an organisms’ genetic information
• Includes the chromosome (either in the cytoplasm of prokaryotes or nucleus of the eukaryotic cell)

Eukaryotic Chromosomes

• Linear, multiple chromosomes
  • Chromosomes: discrete DNA structures with NSL that carry the genes essential for cellular function
  • Located in the nucleus
• Many contain two copies of each chromosome ( diploid ) DNA must be supercoiled to fit inside the cell - DNA packaging proteins: histones - Wrap the proteins up and facilitate its attachment to various scaffolding proteins which help organize the chromosomes - Chromatin Prokaryotic Chromosomes
• Generally circular
• Generally single chromosome within nucleoid region of cytoplasm
  • One copy of of chromosome ( haploid )
• Supercoiled and associated with histone-like packing proteins Extrachromosomal DNA
• Mitochondrial and chloroplast genomes
• Independently maintained viral DNA genomes
• Plasmids
• Small and generally circular pieces of DNA that are found in some bacteria and in some unicellular eukaryotic organisms
• Contain some genes but the genes are not essential for growth
• May provide benefit for growth of the organism under special conditions and could include like virulence factors and antibiotic resistance genes
• Bacteria can exchange the plasmids with other bacteria in a process known as horizontal gene transfer

• “The “gluer”
• Helps glue the DNA fragments together
• DNA replication typically starts at a place called the origin
• Typically, eukaryotes have multiple origins while prokaryotes have only one
• This place is usually identified with certain DNA sequences
• At the origin, the helicase comes in and unwinds the DNA
• In order to prevent the DNA from coming back together so soon, SSB proteins (single-stranded binding proteins) bind on the DNA to keep it separated
• The enzyme topoisomerase keeps the DNA from coiling
• Needs to be controlled during DNA replication
• Can involve an overwinding of the DNA
• Primase comes in and makes RNA primers on both strands for the DNA polymerase to know where to start
• The DNA strands either go tom 5’ to 3’ or 3’ to 5’
• DNA polymerase builds the new strand in the 5’ to 3’ direction
• Means it moves along the old, template strand in the 3’ to 5’ direction
• Adds new bases starting from the 3’ strand
• Old strand is known as the leading strand
• The new strand is known as the lagging strand
• Primers must keep being placed in order for the DNA polymerase to build
• Fragments resulted from this are known to be Okazaki fragments
• Ligase will then glue between the gaps of the okazaki fragments, sealing them together
• At the end, will have two identical helix molecules from the one original
• Semi-conservative
• B/c the two copies contain one original strand and one new strand
• Do not want an incorrect recorded gene that will result in an incorrect protein or no protein
• DNA polymerase has proofreading abilities, rarely making a mistake Summary of Replication Steps in Bacteria - Due to the smaller size of the bacterial genome, DNA replication begins from a single origin of replication and proceeds in both directions around the circular chromosome - Replication occurs in 3 stages: initiation, elongation, and **termination
• Initiation**
• Origin of replication: the site of replication initiation where various proteins bind to being the replication process
• Bacterial chromosomes have one origin of replication
• DNA gyras e (a type of topoisomerase ) relaxes the supercoiled DNA to allow for its unwinding
• Helicase separates the DNA strands by breaking hydrogen bonds between the base pairs. The opening of the DNA creates a Y-shaped structure known as a replication fork. Two replication forks are formed at the origin of replication and move in opposite directions around the chromosome.

• Single-stranded binding proteins bind to the opened DNA strands near the replication form to prevent them from re-annealing.
• DNA polymerase III (the main polymerase involved in replication) needs a free 3' OH end to add new nucleotides to. Primase synthesizes an RNA primer to provide a 3' OH to which DNA polymerase III can add new nucleotides and begin replication. - Elongation
• DNA polymerase III adds nucleotides to the new strand of DNA in the 5' to 3' direction since it can only add new nucleotides to the 3' OH end of a nucleic acid strand. Since DNA strands are antiparallel and run in opposite directions, this leads there being a leading strand that is synthesized continuously in the direction of the opening of the replication fork and a lagging strand that is synthesized discontinuously.
• New RNA primer needs to be constantly added to the growing lagging stand as the replication fork opens up. The lagging strand ends up consisting of many smaller DNA fragments known as Okazaki fragments.
• RNA primers are removed during DNA synthesis by another polymerase called DNA polymerase I. DNA polymerase I replaces the RNA primers with DNA. The gaps between the Okazaki fragments are sealed by the enzyme DNA ligase - Termination
• Less is known about this step
• At the end of bacterial DNA replication the two DNA molecule are interlocked and need to be separated
• With a topoisomerase DNA Replication in Eukaryotic Cells
• As eukaryotic genomes are much larger than prokaryotic genomes, replication proceeds from multiple origins one ach chromosomes
• Major steps of replication are similar between prokaryotes and eukaryotes
• Have different DNA polymerases from bacteria

Transcription 6/

The Central Dogma

- Describes the flow of information in living organisms - Information is stored in DNA and then during gene expression, the information flows from DNA to RNA during transcription and then RNA to protein during translation The Central Dogma and Genotype/Phenotype

• A gene is a segment of a DNA molecule that contains the instructional code that is necessary to direct the synthesis of either a specific protein or a functional RNA molecule (ex: rRNA, tRNA, mRNA)

Initiation

- Promotor - Upstream - Site that lets the transcriptional machinery know that this is where the enzyme needs to bind to and being transcription - RNA polymerase consists of 6 protein subunits - 5 subunits that compose the core enzyme - 6th subunit is known as the sigma factor - Helps the RNA polymerase recognize and bind got the promoter sequence - Once the transcriptional machinery is bond to the promoter sequence, the RNA polymerase has helicase activity - Unwinds the DNA and forms the transcriptional bubble - Transcription is able to proceed Elongation

• After initiation, sigma factors are no longer needed
• Dissociates, but leaves behind the core enzyme complex to complete the transcriptional process
• New ribonucleotides are added to the 3 prime hydroxyl end of the growing RNA molecule
• Complementary to the deoxyribonucleotides of the template DNA strand

Termination

• Self-termination (Rho-independent termination) : transcription of GC-rich terminator region produces a hairpin loop, which creates tension, loosening the group of the polymerase on the DNA - Region of the RNA transcript folds up on itself (hair pin loop) - Creates tension which causes the structure to fall apart
• Rho-dependant termination : Rho pushes between the polymerase and DNA, causing a release of polymerase, RNA transcript, and Rho - Protein - Rho that binds to a sequence in the RNA transcript and then travels down it until it gets to the transcriptional bubble - Causes the polymerase RNA transcript to fall apart Prokaryotic vs. Eukaryotic Transcription
• Differences
• RNA polymerase is different
• Transcription in prokaryotic organisms (like bacteria) take place in the cytoplasm ; in eukaryotic organisms , transcription takes place in the nucleus
• Eukaryotic genes that encode polypeptides consist of coding regions called axons

- Constitutively expressed genes are always transcribed and translated - Genes essential for cell function - Steps - Transcription of the mRNA transcript - Translation of the mRNA transcript to produce proteins - Prokaryotic organisms - Regulation is generally going to happen before transcription - Transcriptional regulation - Eukaryotic organisms - Transcriptional regulation and post-transcriptional regulation - More complex - Operon - Prokaryotic organisms have multiple genes that encode multiple polypeptides of related functions grouped together - Genes that are grouped together in an operon are going to be transcribed together into a single mRNA transcript - As a result, all are under the control of the same regulatory elements - Structural genes that will ultimately be transcribed and translated into proteins - All share one promotor

• RNA polymerase starts at the promoter and and then transcribes all the genes in the operon into a single mRNA transcript - All share regulatory elements
• Operator
• A sequence of DNA that is located in between the promotor and the structural gene site and it can either bind regulatory proteins that facilitate transcription or combine regulatory proteins that block transcription
• Regulatory proteins are encoded by genes somewhere else in the genome
  • Eukaryotic organisms don’t organize their genes into operons Transcriptional Regulation - Tryptophan operon
• One of the amino acids that are found within cells
• Includes several genes that encode enzymes that are involved in tryptophan biosynthesis
• Enzymes that the cell really wants to turn on and off at the same time because they are

all involved in the same biosynthetic pathway, all interact with each other

• Example of repressible operon - Operan that generally is kept on except for when a regulatory molecule becomes present in the cell that represses/shuts down the transcription of the operon - Include genes that are involved in anabolic or biosynthetic pathways - Cell wants to keep the expression of the gene on, except when there is enough of the particular end product accumulates in the cell; then the cell will shut off to conserve more resources and put more resources towards making more end product
• Regulatory protein: trp repressor - When by itself, it is in active form - Cannot bind to the tryptophan operon sequence - When is binded to a molecule of tryptophan, it will active the repressor, allowing it to bind to the operator sequence - When tryptophan is absent in the cell: - The trp repressor dissociates from the operator and RNA synthesis proceeds - Remains in active form - When tryptophan is accumulated in the cell: - The trp repressor binds the operator, and RNA synthesis is blocked - Able to bind to the operator sequence, shut off the tryptophan biosynthesis - Lac operon
• Inducible operon - Operon will generally be shut off, except for when a particular regulatory molecule ( inducer ) becomes present - Typically contain genes involved in catabolic pathways - Generally want to keep them shut off except when the molecule that is being broken down becomes available
• Encodes the genes that are needed for utilizing lactose as a carbon source - When lactose is not present: - Lac repressor will be active and bound to the operator sequence of the operon

• Specific sequences of 3 nucleotides ( codons ) encode a single amino acid
• Genetic code is universal between all living organisms
• 64 possible codon combinations
  • 20 commonly occurring amino acids
    • Redundancy seen is known as degeneracy
• 3 stop codons ( UAA, UAG, UGA ) do not encode any amino acids
  • Tells the ribosome to stop
• “ AUG ” is the start codon
  • Starts the reading frame for polypeptide synthesis
  • Tells the ribosome where to begin amino acid synthesis Mediator Molecule
• Connects mRNA sequence to a specific polypeptide product is tRNA
• RNA molecule that is folded up
• Structure
• At one end, is bound a specific amino acid (ex in picture: glutamic acid)
• At another end, is the anticodon
• Sequence of three bases that will pair with a complementary codon
• When they paid, the glutamic acid (or whatever amino acid is bounded) will be added to the growing polypeptide chain
• Pairing is how the cell knows which amino acid to incorporate at a particular spot in the polypeptide chain Genetic Code
• Starts at the 5’ end and works down to the 3’ end Directionality
• Polypeptides have directionality
• One end: N-terminal
• A free amine group at one end of the polypeptide chain where the other amine groups are binding to adjacent amino acids
• Other end: C-terminal
• A free unbound carboxyl group at that end
• Translation works from the N-terminal of the polypeptide and ends at the C-terminal

Translation machinery - ribosome

• Ribosome is a large complex structure that consists of catalytic RNA, structural RNAs and proteins - 50% of ribosomes are composed of RNA
• One of the key differences that exists between the prokaryotic and eukaryotic translation is that they have different types of ribosomes - Prokaryotes: 70s type of ribosomes - Eukaryotes: 80s type of ribosomes - Difference in ribosome structures allows us to use antibodies to inhibit bacterial ribosomes without inhibiting our own eukaryotic type ribosomes
• Each ribosome (2 subunits)
  • Subunits remain dissociated from each other until after translation initiation has occurred - After translation initiation, mRNA transcript will have the complete ribosomal complex associated with it - Multiple ribosomes will translate along a single mRNA transcript - When one ribosome moves away from the start site, another will be able to hop on - Complex of multiple ribosomes simultaneously translating a single transcript: polyribosome
• Another key difference that exists between prokaryotic and eukaryotic translation is that prokaryotic translation happens simultaneously to transcription - Because they both occur in the cytoplasm since they do not possess organelles - Once the RNA polymerase and starts adding some codons, the ribosome can hop on the the mRNA transcript at that point - In eukaryotic organisms, they occur physically and temporally separate - Translation occurs outside of the nucleus

Elongation

• Initiator tRNA molecule did enter at the P site
  • Now, every other charged molecule will now begin to enter at the A site
• Second codon of the mRNA transcript in the A site
  • Second tRNA that is charged will enter the A site - It’s anticodon will pair with te compliment final codon in the mRNA transcript
• Peptide bond
  • Bond that links together amino acids within a polypeptide chain
• At this point, fMet has become detached from its carrier tRNA and now is bound via. Polypeptide bond to the Phe which is still bound to the tRNA molecule - Whole complex will shift one spot over - Now will have an uncharged tRNA molecule in the exit site (E site) and then have the tRNA carrying the growing polypeptide chain in the P site - 3rd codon is now exposed in the now empty A site Termination
• Once a stop codon appears within the A-site, this triggers the termination of translation
• Instead of a charged tRNA molecule binding to that codon, a protein known as a release factor will come into the A-site and bind to the stop codon

• Binding of release factor: triggers the dissociation of the entire complex so forest the large subunits fall apart and then also the newly synthesized polypeptide is released as a free polypeptide - Energy-dependent step that requires GTP Difference between Eukaryotic and Prokaryotic transition
• The mechanisms of how you know the ribosome moves down the mRNA and how the polypeptide chain is synthesized is essentially the same between eukaryotes and prokaryotes
• Prokaryotes: transcription and translation occur simultaneously within the cytoplasm because that is where the prokaryotic chromosomes are located since there are no organelles
• Eukaryotes: transcription occurs in the nucleus because that’s where the DNA is located and the mRNA transcript that is produced will be processed into a mature mRNA transcript which then will get exported into the cytoplasm and there the mRNA transcript will either be translated by the free ribosomes within the cytoplasm or by the ribosomes associated with the rough ER