PBL 1: The Blueprint of Life

DNA

a genetic molecule that carries the genetic blueprint for the development, functioning, growth and reproduction of all living organisms

Double Helix Structure

two polynucleotide chains (complementary strands because of base pairing) wrapped around each other

How are there other types of DNA?

other secondary structures can occur due to conditions such as the nature of the positive ion associated with the DNA and the specific sequence of bases

A-DNA and Z-DNA

• A-DNA forms under low humidity or high salt conditions, and its shorter, more compact and wider

• Z-DNA may form temporarily during active transcription. As RNA polymerase moves forward, DNA behind it becomes negatively supercoiled. Certain sequences can switch from right-handed B-DNA to left-handed Z-DNA, helping relieve some torsional strain. When the strain disappears, Z-DNA usually returns to B-DNA.

Base Stacking

hydrophobic bonding (van der Waals connections) that occur between bases on the same DNA strand

Topoisomerase Class I and II

• Topoisomers: relieves straining that is due to supercoiling

• Class I: cuts the phosphodiester backbone of one strand of DNA, passes the other end through and reseals

• Class II: cuts both phosphodiester backbones of DNA, passes some of the remaining DNA helix between the cut ends and reseals

Nucleophile

in DNA replication the 3’ OH group of sugar acts as a nucleophile and attacks the phosphate group on an incoming nucleotide. this nucleophilic attack creates a phosphodiester bond

Primer

• Short oligonucleotide strand to which the growing polynucleotide chain is covalently attached to in the early stages of replication

• Hydrogen bonded to template strand and made up of ATP, UTP, GTP and CTP (ribonucleoside triphosphates (monomer of RNA)

Replisome

multi-protein complex that works together as a single machine to copy DNA

DNA Gyrase

type of topoisomerase (II) that works using ATP to relieve straining ahead of the replication fork due to unwound DNA

SSBPs

Stabilises the single stranded regions by binding tightly

Primosome

a multi protein complex that includes primase and helicase to organises and activase primer synthesis

Difference between DNA replication in Prok and Euk

• Prok: no proteins complexed to DNA, Okazaki fragments 1000-2000 residues long, one origin of replication, polymerase are all exonucleases

• Euk: histones complexed to DNA, Okazaki fragments 150-200 residues, multiple origin of replication, not all polymerases are exonucleases

DNA Poly I, II, III, IV and V

I: proofreads DNA sequences and removes RNA primers and replaces with DNA nucleotides (repairs, proofreads, synthesises and removes)

II: repair enzyme

III: main polymerise enzyme

IV and V: repair enzymes under unusual conditions

DNA Poly (alpha, beta, gamma, delta, epsilon)

DNA polymerase α: starts replication by working with primase to make a short RNA primer followed by a short stretch of DNA.

Pol β: DNA repair

DNA polymerase ε: mainly synthesizes the leading strand continuously.

DNA polymerase δ: mainly synthesizes the lagging strand as Okazaki fragments.

• delta

DNA polymerase γ: replicates mitochondrial DNA.

Differences between DNA and RNA

Presense of 2’ hydroxyl group, uracil and thymine, RNA has ribozymes

Why is RNA good for transcription and translation

the extra 2’ OH makes RNA unstable, which leads to an auto-cleavage reaction. This is good because mRNA must quickly be translated or it will be degraded, and if mRNA were stable it would continue to occur well after a gene has been transcribed even if cellular conditions no longer require that protein

Uracil Characteristics

short-lived, continuously produced and replaced and not in the cells permanent genetic archive, therefore mutation in RNA molecule is less serious than permanent mutation in DNA

Ribozymes

RNA forms catalytic molecules that can catalyse biochemical reactions. They coordinate with metal ions to fold into complex active shapes through interactions occurring at distant parts of the molecules

• DNA doesnt have them because it prioritises stable information storage of chemical reactivity

Ribosomal RNA

sites for assembly of growing polypeptide and forms the core structure of ribosomes

Transfer RNA

Transports amino acids to the site of protein synthesis

a tRNA is a single polynucleotide chain between 73 and 94 nucleotide residues long

mRNA

Carries mature RNA from the nucleus to the cytoplasm/ribosomes used as gneetic instructions for protein synthesis

New Age RNA

Small Nuclear: processing initial RNA into mature RNA for export

Micro: bind to phage DNA to prevent infection and repairs nerve damage

• also can bind to mRNA and activate or inhibit processes depending on which mRNA it’s bound to

Small Interfering: used to eliminate undesirable genes (like uncontrollable cell growth)

Why do the core enzyme and holoenzyme have to work together?

The core enzyme canpolymerise a new RNA strand however it lacks specificitiy and therefore would synthesise both DNA strands. The sigma subunit helps bind the RNAP to the correct promoters and then dissaciates after 10 NT

Consensus Sequences and their Function

Many of the base sequences in different promoter regions of prokaryotes are similar

• Binding RNA polymerase and the frequency with which the gene needs to be transcribed (strong promotor=more frequent)

DNA Scrunching

Because the sigma subunit is tightly bound to the promoter, the RNA polymerase is fixed, therefore it pulls DNA into the transcription complex and the unwound DNA creates torsional stress

• Sufficient Energy: RNA polymerase breaks promoter interactions and moves toward elongation

• Insufficient: abortive transcription, the short RNA is released

Different Types of RNA Polymerase (EUK)

• I: found in nucleolus and synthesises most precursors of rRNA

• II: found in nucleoplasm and synthesises mRNA precursors

• III: found in nucleoplasm and synthesises tRNA precursors, 5S rRNA, and a variety of other small molecules involved in mRNA processing and protein transport

Split Genes

Genes whos coding instructions are broken into coding sequences (exons) and non coding sequences that are removed (introns)

Splicesomes Structure and Function

made up of multiple small nuclear ribonucleoprotein particles (snRNPs) (snRNA and proteins found in the nucleus that remove intons

snRNPs and SPlicesomes are needed to mediate the process

What is needed splicing

GU at 5’ end (part of intron), AG at 3’ end (part of intron) and a branch site containing an A that can perform a nucleophilic attack on 5’ GU

Splicing Process

The free 3′-OH of exon 1 attacks the phosphate at the 3′ splice site (AG) which creates a phosphodiester bond between exon 1 and 2

Isoforms (Alt Splicing)

One gene can create multiple forms of proteins through different exon combinations (leads to different amino acid sequences and therefore different proteins)

Prokaryotic Regulation of Transcription

• Alt Sigma Factors: Sigma factors decide which genes through telling RNA polymerase which promotor to recognise, therefore changing different sigma factor = different gene transcribed

• Enhancers: DNA sequences that decide the frequency at which the gene is transcribed

  • Provide transcription factors which are binding sites for regulatory proteins

• Operons: structural genese, promoter and operator region

  • Repressor and Inducer binding to Operator

• Transcription Attenuations: determine whether RNA polymerase should continue or stop depending on the structure

  • In Prok transc and transl occur at the same time, and the mRNA folding influences:

  • If ribosome moves quickly, termination structure may form and stops

  • If ribosome stalls, alternative structure may form and continues