BIOC221

Prokaryotic genes

Smaller, circular, fewer genes, lack introns, organised as operons.

Eukaryotic genes

Larger, linear, contain introns and complex regulatory sequences

- Have histone proteins for DNA packaging

Promoter

A regulatory region where RNA polymerase binds to initiate transcription

Exons

Coding regions that are transcribed and translated into protein

- remains in mRNA after splicing

Introns

Non-coding regions that are transcribed but spliced out before translation

- removed from pre-mRNA during splicing

5'UTR

The 5' Untranslated Region of mRNA is important in initiation and regulation.

3'UTR

(Untranslated region) a region of mRNA between the stop codon and the 3' end. Involved in mRNA stability and translation regulation.

Splicing

Removal of introns from pre-mRNA to produce mature mRNA.

Capping

Addition of a 5' cap to mRNA for stability and recognition by ribosomes.

Polyadenylation:

Addition of a polyA tail to the 3' end of mRNA, enhancing stability and translation.

How is DNA packaged in the nucleus?

DNA is wrapped around histone proteins to form nucleosomes. Nucleosomes coil and fold to form chromatin fibers.

Euchromatin

loosely packed chromatin

- transcriptionally active

- can switch between active and inactive

Heterochromatin

highly condensed chromatin

- inactive

Different roles for enzymes in the replication fork:

DNA Helicase: unwinds DNA helix

Single-Strand Binding Proteins (SSBs): Prevents DNA strands from reannealing

DNA Primase: Synthesizes RNA primers

DNA Polymerase: Synthesizes new DNA strands

DNA Ligase: Seals nicks in the DNA backbones

Topoisomerase: Reduces supercoiling

Leading Strand:

Synthesized continously in the 5' to 3' direction

Lagging Strand:

A discontinuously synthesized DNA strand that elongates by Okazaki fragments, requiring multiple RNA primers

- DNA polymerase repeadetly dissociates and associates after syntheisizng each fragment

Making cDNA from RNA:

using the enzyme Reverse Transcriptase

- Reverse transcription to synthesize a DNA strand using RNA as a template

- Degradation of the RNA template by RNase H

- Second strand synthesis using DNA polymerase

Define Bioinformatics

The development and application of computer science to the analysis of large amounts of biological information

How is a nucleotide incorporated into DNA by DNA polymerase.

DNA polymerase adds nucleotides to the 3′ OH end, using dNTPs that pair with the template strand (A=T, C≡G), building the new strand 5′ → 3′

DNA helicase

unwinds the DNA helix

Single-strand Binding Proteins (SSBs)

prevents DNA strands from reannealing

- in Replication fork

DNA Primase

Synthesizes RNA primers for DNA replication in the replication fork

DNA Polymerase

Synthesizes new DNA strands

- Replication fork

DNA Ligase

Seals nicks in the DNA backbone

- Replication fork

Topoisomerase

Reduces supercoiling

- replication fork

Prokaryotic versus Eukaryotic mechanisms for DNA replication

Prokaryotic:

- has a single origin (oriC)

- use Pol III for replication

Eukaryotic:

- have multiple origins

- use a combo of Pol α, δ, and ε

- involves telomere maitenance

Understand how DNA replication initiates & terminates in Prokaryotes

Initation: DnaA binds to oriC to begin replication

Termination: Tus proteins bind to Ter sites to prevent fork progression

Understand how DNA replication initiates & terminates in Eukaryotes

Initation: ORC binds to chromatin before the S phase, loading MCM helicase

Termination: Telomeres at chromosome ends are maintained by telomerase

Recognise the process involving RNA template to maintain telomeres

Telomerase extends the telomeres by adding repetitive DNA sequences using an RNA template, therefore preventing chromosome shortening

What happens when the DNA replication process goes awry?

Errors in DNA replication can lead to mutations, genome instability, cancer, and developmental disorders such as primordial dwarfism

Understand how PCR works and can be used to amplify DNA

- PCR (Polymerase Chain Reaction) amplifies specific DNA sequences using primers, dNTPs, DNA polymerase and temperature cycling

- The process involves denaturation (95 degrees C), annealing (50-67 degrees C), and extension (67-72 Degrees C) steps repeated in cycles to expontenially amplify DNA

Review DNA structure and know what a dideoxynucleotide is:

DNA structure: Made up of nucleotides

- phosphate group

- nitrogenous base

- deoxyribose sugar

ddNTP: Didepxynucleotide:

- lacks a 3' OH group, preventing further DNA extension when incorporated, critical in Sanger Sequencing.

How does Sanger sequencing work?

ddNTP terminates chain, making fragments of different lengths, these can be separated by gel electrophoresis

- Fluorescent tags attatched to ddNTPS allow the sequence to be determined

Key concepts of Next-generation sequencing

Steps in cDNA synthesis

1. Primer is added to RNA (often pol T)

2. Reverse Transcriptase synthesises a single DNA strand

3. RNA strand is degraded by RNase H

4. DNA polymerase synthesizes second DNA strand, forming double-stranded cDNA

Outline the steps in quantitative PCR

qPCR measures DNA amplification in real time using fluorescent signals such as SYBR Green or TaqMan Probes

- You get a Ct value, indicating the point at which fluorescence rises above the baseline, allowing quantification

Quantification involves comparing gene expression to a housekeeping gene

Outline the steps in RNA-seq

1. Use reverse transcriptase to convert RNA to cDNA

2. cDNA is fragmented into smaller pieces, create sequencing library

3. Use next-gen sequencer

4. Sequence reads are alligned to a reference genome

What does RNA-Seq tell you?

- Transcrip level analysis

- Quantification

- Alternative splicing

- Novel transcripts

What is comparative sequence analysis?

Comparative sequence analysis compares sequences across different species to identify conserved regions, which may indicate functional importance. Sequence similarity often suggests a common ancestral origin.

How are molecular biology tools useful in investigating genomic differences?

PCR, Sanger sequencing, next-gen sequencing and CRISPR are essential in studying genomic differences

- These tools help identify disease causing mutations, track inheritance patterns & study gene function

Define bioinformatics role in Molecular Biology

provides essential infrastructure, ways to store and access data

BLAST:

- Basic Local Alignment Search Tool

- compares a DNA or protein sequence to data bases to find regions of similarity, helping identify gene functions or evolutionary relationships

InterProScan:

Identifies protein domains and functional sites by comparing known domain databases to sequences

AlphaFold:

Artificial intelligence system capable of predicting 3D protein structure directly from amino acid sequencing

AlphaMissense

Predicts how a single amino acid change affects protein function. Helps assess impact of genetic variants

Examples in which Bioinformatics and Molecular Biology contribute to addressing current questions

- Medicine: identifying pathogenic variants using Clinvar or AlphaMissense

- COVID-19: monitoring viral genome changes using BLAST and sequence analysis

- Climate change: reduce methane emissions by analysing microbial genomes and developing vaccines

Outline why the location of related genes in operons is important for gene expression in bacteria

- Ensures coordinated expression

all genes in operon are turned off/on together, so proteins are made at the same time in equal amounts

- Efficiency

Grouping genes allows to turn on a whole process in one step

- Simplifies regulation using shared control elements

Operon

A set of co-transcribed genes under the control of a single regulated promoter

Outline why the regulation of gene expression is important in prokaryotes

- Saves energy: only express genes when necessary

- Responds to changes in environment: allows genes to adapt quickly

- Ensures coordinated gene control: Bacteria coordinate entire pathways by turning on/off groups of genes

The CRISPR-Cas system as an example of a bacterial noncoding RNA and RNA-protein complex

The CRISPR-Cas system is a bacterial defense system made of:

Noncoding RNA (crRNA)

That forms a complex with Cas proteins→ Together, they find and cut viral DNA to protect the bacterium

Key features of bacterial promoters

–35 region → –10 region → Initiation site +1 = Recruit → Unwind → Begin

Describe the structure of RNA Polymerase in bacterial transcription

2 alpha subunits = enzyme assembly, promoter recognition

B subunit = catalytic activity

B' subunit ^

sigma factor = promoter specificity

Describe the role of RNA polymerase in bacterial transcription: INITIATION

- RNA Pol Holoenzyme binds to promoter region

- Alpha helices of sigma factors recognise -10 and -35 regions on promoter

- DNA unwinds to form open complex

- after a few nucleotides are added, sigma factors detach

Describe the role of RNA polymerase in bacterial transcription: Elongation

- core enzyme synthesizes RNA in the 5' to 3' direction

- Beta subunit catalyzes phosphodiester bonds

Outline an example the mode of action of antibiotics that inhibit bacterial transcription

Rifampicin binds to the beta subunit of RNA polymerase which inhibits transcription

Rho-dependent transcription termination

Rho protein recognizes specific DNA sequences and causes a pause in the RNA polymerase

Rho-independent transcription termination

Transcribed mRNA forms a hairpin (stem) loop followed by multiple U nucleotides → Causes a pause in RNAP

What does the lac operon involve?

- LacZ, LacY, lacA genes

- Promoter (where RNAP binds)

- Operator (where the repressor can bind and block transcription)

- Lacl (codes for Lac Repressor)

- CAP site (where CAP-cAMP binds to boost transcription when glucose is low

What's the trp operon?

A biosynthetic operon containing genes encoding enzymes to make Tryptophan (Trp) an essential amino acid

- Enzymes are not made if tryptophan is already available

Feedback loop 1: Trp operon

- Trp levels high, Trp acts as a corepressor

- Binds to TrpR (repressor), changing its shape

- TrpR binds to operator region of operon

- RNAP blocked no transcription

Trp operon Feedback loop 2: Attenuation

- found in leader sequence

- leader mRNA can fold into secondary structures (hairpins) based on how fast the ribosome moves

The importance of comparative sequence analysis:

- Compares sequences across different species to identify conserved regions; the consevered regions indicating a functional importance

- Sequence similarity suggests a common acestral origin

lac vs. trp Operon Regulation

Lac operon:

- breaks down lactose for energy

- catabolic

- inducible

- Allolactose = inducer

Trp operon:

- synthesizes trypotophan

- biosynthetic

- Repressible - turned off by Trp

Tryptophan = corepressor

RNA Polymerase I

- Located in the nucleolus

- Synthesizes rRNA

RNA Polymerase II

- Located in the nucleoplasm

- makes; mRNA, snoRNA, snRNA, miRNA, lncRNA

the workhouse for making RNAs involved in gene expression

RNA Polymerase III

- Located in the nucleoplasm

- Transcribes tRNAs, 5S rRNA, and some snRNAs

Main types of non-coding genes

- rRNA (ribosomal RNA)

- tRNA (transfer RNA)

- snRNA (small nuclear RNA)

- snoRNA (small nucleolar RNA)

- miRNA (micro RNA)

- lncRNA (long non-coding RNA)

What is meant by gene expression

The process by which the information in a gene is used to make a functional protein

Alterations in DNA

Variants in DNA can cause differences in gene function, leading to genetic disorders or variations in traits

- e.g. a vairant in the CFTR gene -> causes cystic fibrosis

DNA structure:

DNA is a double-stranded, antiparallel helix with a sugar-phosphate backbone and complementary base pairs (A-T and G-C) held together by hydrogen bonds

ddNTP

- lacks a hydroxyl group (OH) at 3'

- prevents further DNA extension when incorporated

- critical in Sanger sequencing

Maintaining telomeres with an RNA template

- Telomerase extends the telomeres by adding repetitive DNA sequences using an RNA template, preventing chromosome shortening

The basic structure of eukaryotic RNA Polymerase II

Multi-subunit enzyme:

Rpb1: catalytic: contains CTD important for regulation and RNA processing

Rbp2: helps catalyse RNA synthesis

Eukaryotic specific subunits: regulatory roles, chromatin interaction

Why does RNA polymerase need assistance to recognize and bind to DNA, particularly when the DNA is packaged into chromatin?

Nucleosome remodellers

Use ATP to move, eject, or restructure nucleosomes , making DNA more or less accessible for transcription function

Structure of a protein-coding gene

- Transcription start site

- Core Promoter

- Cis-regulatory elements (CRE)

Spliceosome structure

- snRNPs

- dynamic

- requires ATP to assemble and rearrange

Spliceosome role

- Catalyzes mRNA splicing (removes introns, joins exons together

- Performs two transesterification reactions

- Ensures accurate and efficient mRNA processing befote export

How is chromatin modified during transcription?

P-TEFb (positive transcription elongation factor b) role:

helps transition to productive elongation

Phosphorylates:

- The CTD of RNA Pol II at Ser 2

- The negative elongation factors

this releases the pause and lets RNA Pol II enter productive elongation

Why is P-TEFb important?

- Without P-TEFb, transcription would stall early

- P-EFTb is the gatekeeper to real elongation

- Productive elongation = mature mRNA production

What is the 5' capping?

the addition of 7-methyl guanosine to the 5' end of mRNA

Formation of the Pre-Initiation Complex (PIC)

- TFIID binds first at the core promoter e.g. TATA box

- Other GTF (General Transcription Factors)

- RNA Pol II is recruited

- TFIIH unwinds DNA and phosphorylates the CTD of RNA Pol II

- Phosphorylation critical for initiation of transcription

Steps in Activator-Dependent Recruitment of RNA Pol II

1. Transcription Factor binding

activators bind to enhancer DNA sequences and holds activation domain

2. Recruitment of Co-activators

co-activators help with protein-protein interactions and modify chromatin

3. Chromatin remodeling at the Core Promoter

histone tail modifications, nucleosome remodelling, Core Promoter = accessible,

4. Formation of the PIC

TFIIH binds at core promoter, RNA Pol II recuited, TFIIH unwinds DNA, phosphorylates CTD

CTD of Rpb1 subunit of RNA Pol II

- Unstructured (floppy) and non-catalytic

- Made of heptapeptide repeats (7 Amino Acid)

- Reverlibly phosphorylayted during transcription

Epigenetics

the study of influences on gene expression that occur without a DNA change

Describe the Histone code

describes the series of modifications on histone tails that contribute to controlling whether DNA can be transcribed

Describe the histone codes effect on transcription

Describe the features of tRNA

- Small and folded

- 4 key loops

- Modified bases

Describe the function of tRNA

adaptors to translate nucleotide sequence to amino acid sequence

- important for protein synthesis for translation

- cloverleaf secondary structure

Describe the features of the ribosome

- scaffold and catalyst for translation

important to link together amino acids

Describe the mechanism for coupling an amino acid to a tRNA

What are Isoacceptor tRNAs?

tRNAs that have different anticodons but carry the same amino acid

- recognise different codons for the same amino acid

Understand the importance of codons and anticodons in protein synthesis

- Codons (in mRNA) and anticodons (in tRNA) pair specifically during translation

- This pairing ensures the genetic code is translated correctly into a protein

- Accuracy in this pairing is essential for functional proteins and proper cell function

What is Sanger Sequencing used for?

•Sequence individual genes

•Verifying the presence of a specific DNA fragment

Name two features that differentiate an expression vector from a cloning vector

Promoter: Expression vectors possess a promoter sequence to drive transcription of the cloned gene, which is usually absent in cloning vectors.

Ribosome Binding Site (RBS): Expression vectors include an RBS to facilitate translation of mRNA into protein, ensuring the cloned gene is translated.

RT-qPCR vs RNA seq

RT-qPCR is used for: validating RNA-Seq data, studying expression of specific genes, diagnostics

RNA seq is used for a comprehensive measurement of gene expression, allowing detection of changes in expression of ALL genes

RNA-seq is a discovery tool; RT-qPCR is a validation tool.

Describe an experiment you can use to confirm that the FAST ORF has been successfully cloned into the pCRII cloning vector

- Restriction enzyme digest

- Perform PCR

- Sanger sequencing

What is the purpose of feedback inhibition in biosynthetic pathways?

To prevent overproduction of metabolites

What do sigma factors do?

Recognise specific promoter sequences + makes RNA Pol bind to the correct location on DNA