BIOL 220Describe the central dogma and identify cis-acting and trans-acting components of transcription

L7 - Describe the central dogma

Central Dogma: DNA (transcription) → RNA (translation) → Protein

L7 - Identify trans- and cis-acting components of the transcriptional machinery

Cis-acting: DNA sequences (promoters, TATA box, enhancers, silencers, operators)

Trans-acting: Proteins or other diffusible factors (transcription factors, TATA-box binding proteins)

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Cis-acting: confer specificity at the level of the genes

Trans-acting: confer specificity at the level of the cells

L7 - Define how cis sequences control which strand will be transcribed

Promoter: DNA sequence near the start site where RNA polymerase starts transcription — also recruits and positions transcription factors — upstream of transcription start site

Bacteria: Consensus sequences (-10 and -35) — RNA poly sigma factor recognizes these

Eukaryotes: Consensus sequences (TATA, CAAT, GC-rich) — different combinations

• Consensus sequences is a pattern shared by many related DNA sequences — the exact sequence can vary but certain positions are more conserved (required to work)

Mutations in sequence → less transcription means position is important to promoter function

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The promoter’s orientation and polarity determines which direction transcription proceeds and therefore which DNA strand serves as the template (will be read 3 → 5 to be built 5 → 3)

L7 - Describe the events taking place during transcription

Initiation: Transcription factors and RNA polymerase assemble at the promoter, forming the pre-initiation complex near the transcription start site

• TFIIH joins the complex and acts as a helicase (unwinding DNA) creating a transcription bubble + first RNA nucleotides added by RNA polymerase

• A kinase subunit of TFIIH (CDK7 / cyclin H) phosphorylates the tail of RNA poly II to transition into elongation (w/o kinase, no transition into elongation = end after initiation)

Elongation: RNA polymerase continues synthesizing the RNA 5’ → 3’

• Protein kinase, a positive transcription elongation factor (P-TEFb) helps phosphorylate 5→3

Termination: the end of transcription once the polymerase reaches the end of coding sequence

Bacteria: Intrinsic termination and Rho-dependent termination

• Intrinsic termination: The RNA forms a hairpin from complementary sequences followed by multiple U-residues (cis-acting)

• Rho-dependent: The Rho protein helps separate the RNA from the transcription complex when polymerase stalls (a bunch of G-residues) → Rho pulls mRNA away from polymerase

Eukaryotes: Lack termination sequences, instead have RNA processing → Splicing

L7- Synthesize and summarize the mechanisms and outcomes of RNA processing

The 5’ Cap: G-residues added onto the 5’ end of the RNA — Capping enzyme rides on the polymerase II and as soon as the 5’ end of the transcript occurs, the cap starts

• Protects from degradtion

• Facilitates transport of mRNA to the cytoplasm

• Target the mRNA to the ribosome

The 3’ poly-A-tail: Cleavage and polyadenylation specificity factor (CPSF) recognizes polyadenylation signal (AAUAAA) and helps cleave the RNA shortly after → the poly-A-polymerase adds about 250 A residues to the new 3’ end

• Protects from mRNA degradation

• Facilitates transport to cytoplasm

RNA splicing: Spliceosome, a complex containing proteins and small RNAs, recognizes splice-relates sequences and brings the appropriate RNA regions together so that introns can be removed and exons can be ligated together (GU —- AG)

L7 - Predict the functions of cis and trans elements in regulating alternative splicing

Exonic splining enhancer motifs (ESEs) in the mRNA (cis) and RNA binding proteins in the SR family (trans) facilitate correct splice binding recognition

• If one is mutated, splice site recognition would be altered causing the genetic code in the mRNA to be messed up

L7 - Given summary

• The transcriptional machinery binds to promoter motifs

• RNA polymerase acts with transcription factors

• RNA synthesizes RNA 5’ → 3’

• In bacteria a termination sequence terminates transcription

• pre-RNA is processed to generate mRNA

L9 - Describe how peptide bonds assemble amino acids into proteins

Amino Acid: amino group, carboxyl group, central carbon, and variable R-group

• a peptide bond forms between the carboxyl group of one amino acid and the amino of another + hydrolysis = dipeptide

• N-terminus = amino group = NH2 (first) & C-terminus = carboxyl group (last) = CHO2

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Initiation: the small ribosomal subunit binds the 5’ cap and scans the mRNA in the 5→3 for AUG

• first tRNA is recruited into the P-site

Ribosome Assembles: the large subunit joins

• second tRNA enters the A-site, matching it’s anti-codon to the next

Elongation: The ribosome catalyzes peptide bond formation

• growing protein is transferred to the tRNA in the A-site and ribosome advances by a codon

tRNA Movement: ribosome moves along the mRNA, tRNA moves through the A→P→E sites

• new tRNAs continue to move through A-site

Termination: ribosome reaches a stop codon

• release factor promotes release of the completed protein + ribosome disassembles

L9 - Explain how tRNA molecules function as interpreters of the language of RNA to protein

tRNA: acts as an interpreter between the mRNA codons and the amino acids used to build a protein

• Anti-codon: a sequence of 3 nucleotides that base-pairs with a complementary codon on the mRNA → recognizes the appropriate mRNA codon

• Amino acid attachment site: other end where the tRNA carries a particular amino acid

example: mRNA codon = 5-UUC-3 → tRNA anticodon = 3-AAG-5 → amino acid: Phe

• enzyme: Aminoacyl-tRNA synthetase attaches the appropriate amino acid to it’s matching tRNA → super specific = isoaccepting tRNA

Stop codons: UAA, UAG, and UGA

• Wobble pairing = the first 2 bases of a codon are important, but the 3rd is flexible (pg. 47)

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Transcription start site does NOT equal translation start site

A small ribosomal subunit binds near the 5’ cap and scans in the 5→3 direction looking for AUG

• AUG starts a reading frame = a stretch of codons in one reading frame that begins with a start and ends with a stop codon — correct one is the longest one

L10 - Explain how the lac operon regulates gene expression in response to lactose and glucose availability

Lac operon: on or off control switch for lactose by allowing or not allowing repressor to bind

• Beta-galactoisdase (lacZ): Breaks lactose into glucose and galactose

• Lactose Permease (lacY): Helps transport lactose into the cell

• Galactoside Acetyltransferase (lacA): still unknown role

• lacI: repressor gene

lacI ——- Promoter—Operator—lacZ—lacY—lacA

• RNA polymerase binds to the promoters and transcribes the operon, lac Z, Y, and A

Inducible Operon: without lactose present, transcription is blocked/repressed

• when lactose is present, the lactose binds to the repressor and removes it = transcription on

CAP is a regulated transcriptional activator that recruits RNA poly to turn on genes w/o glucose

L10 - Predict the effects of mutations in the lac operon, including changes to the promoter, operator, repressor, and lacZ gene

NO lactose + glucose = low lacZ activity

lactose + NO glucose = high lacZ activity

Removing promoter = low lacZ activity

Removing operator = high lacZ activity (repressor cannot bind)

Remove lacI = high lacZ activity (no repressor made)

• Constitutive = mutant proteins that are always active without the usual upstream signal

lacI gene is TRANS-acting so if gene is moved then it can still block transcription

L10 - Describe bacterial conjugation and explain how plasmids can transfer DNA, including antibiotic-resistance genes, between bacteria.

F-plasmids: can replicate within host cell + partition to daughter cells — Bacterial Conjugation:

1. Cells connect: F(+) carries plasmid builds a pilus that connects to a F(-) recipient

2. F-plasmid is nicked and one strand is transferred through pilus to F(-)

3. - Helicase is called TraI

4. Host DNA polymerase replicates the second strand in each cell so now both have double stranded F-plasmids = now both F(+)

Transferring antibiotic resistant among bacteria

L11 - Explain how tissues can express different genes from the same genome

Transcription factor: is a protein that binds specific DNA sequences and helps regulate transcription

• Different cell types express different transcription factors (same genome, different factors)

Promoter: is a DNA sequence where RNA polymerase and associated general transcription factors assemble to initiate transcription

Enhancer: is a regulatory DNA sequence that helps increase transcription when the appropriate transcriptional activators interact with it

• Can be far away from promoter sequence because it can loop the DNA to bring transcription factors near it → cohesion + CTCF binding sites

Silencer: is a DNA sequence that can reduce transcription when the appropriate transcriptional repressors bind to it

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Transcription Factor Mutations: Microthalmia + Cleft palate

• Microthalmia (smaller eyes): heterozygous, LOF, Homeodomain, TF: Otx2

• Cleft palate: heterozygous, LOF, HLH, TF: IRF6

Haploinsufficiency: half as much protein is NOT enough, so homozygous is lethal

L11 - Compare and contrast transcriptional regulation in prokaryotes and eukaryotes

L12- Define epigenetics and explain how epigenetic mechanisms regulate transcription

Epigenetics: A heritable phenotypic alteration in which the DNA sequence is unchanged

• resulting from distinct chromatin states affecting transcription in specific types of cells

• heritable = inherited from a cell to its daughter cells

DNA Methylation: involves adding a methyl group to a DNA base

• occurs next to guanine in a CpG site → 5mCpG3

• many next to each other = CpG islands

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Methylation correlates to gene expression

• the more methylated the gene is, the less it is expressed

• the more expressed the gene is, the less methylated it is

Each cell types has a unique methylation profile that is established after MOST methylation is erased in both sperm and eggs

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How DNA methylation is heritable:

1. Before replication: both strands are methylated at the relevant sites

2. After replication: each DNA copy contains an old methylated strand and a newly unmethylated strand → hemimethylated

3. Dnmt1 adds methyl groups to the newly synthesized strands

4. Methylation pattern is maintained → the daughter cells can retain the same gene-silencing

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Testing methylation to see if it really silences genes:

• 5-azacytosine can be incorporated in replication but cannot be methylated

• many genes that had been OFF turned ON

• long-term killed cells, but short-term changed cell fate

L12 - Explain genomic imprinting and predict outcomes of mutations in imprinted genes

Genomic imprinting: the expression of a copy depends on whether it came from the mother or the father because you inherit two copies of a gene, one from each parent but for some the expressions depends on which parent expresses what\

• H19 and IGF2: Mother’s H19 was not methylated, and dad’s IGF2 was not methylated, causing both genes to be expressed — if one were to mutate, the other parent’s genome could not make up for it because their other gene was methylated

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DNA within cells is NOT naked: DNA + histones = Chromatin

Histones caused decreased gene activity because the DNA is tightly wrapped around the histone protein and cannot be accessed by transcription factors

• However, there are histone tails that stick out (short regions of protein coding sequences)

Post-translational modifications: Acetylation and methylation

• Acetylation: promotes gene activity → w/o acetylation = gene becomes inactive (X)

• Methylation: decreases gene activity

Writers: Adds modifications (methyltransferases + acetyltransferases)

Erasers: Deletes modifications (deacetylases + demethylases)

Readers: Use protein domains that bind particular histone modifications (chromodomains)

L13 - Explain the role of alternative splicing in gene regulation, and how it is regulated

One gene can produce multiple genes through alternative splicing - Different combinations of exons are retained to differentiate between cell types

• Spliceosome recognizes splice donor and splice acceptor sequences but can use different combinations of splice sites

• Splice donor is 5’ and acceptor is 3’

• Each exon has multiple alternative within it, so choosing one from each chosen exon creates further combinations of genes

RNA binding proteins in the: SR protein family bind to sequences in exons and direct the spliceosome to include or exclude exons — by either enhancing or inhibiting so they are skipped

• ESE = exonic splicing enhancer motifs: provides the binding site for inclusion/exclusion

• Different sets of SR proteins are expressed in different cells

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Spinal muscular atrophy (SMA): affects the motor nerve cells in walking, breathing, eating, etc.

• SMN gene was duplicated and one has a mutation that alters splicing

• In the 7th exon, the C → T, causing the exon to be skipped in splicing and becomes not functional (cis-acting mutation affecting trans-acting element)

• As long as the first SMN gene is intact then the individual is fine — if there is a homozygous mutation then the individual has SMA because SMN 2 cannot make up for 1

• Anti-sense oligonucleotides can correct splicing and alleviate symptoms

L13 - Explain the functions of miRNAs in regulation

miRNA: is a small functional RNA that regulates a target mRNA by binding to a complementary sequence (3’ UTR) → lin-4

C. Elegans: lin-14 promotes transcription (repeat L1) and lin-4 inhibits transcription (skip L1)

• lin-4 is complementary to the 3’ UTR of lin-14 — inhibits gene expression of lin-14 mRNA by binding to the 3’ UTR — by deleting lin-14 UTR, lin-4 could not bind and expression increased

• lin-4 does not have ORF but does encode for functional RNA

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1. pri-miRNA: the miRNA gene is transcribed into a long primary RNA transcript

2. Drosha: A protein complex containing the RNase Drosha trims the pri-miRNA into a hairpin-shaped pre-miRNA of ~ 70 nucleotides

3. Cytoplasm + Dicer: now in the cytoplasm, the Dicer that recognizes double stranded RNA cleaves off the terminal loop of the hairpin and cuts RNA to ~ 22nt miRNA duplex

4. Argonaute and RISC: The duplex associates with AGO. The passenger strand separates, leaving the guide strand in the RNA-induced silencing complex (RISC)

5. Target mRNA: the guide strand directs RISC to an mRNA with a complementary target sequence, leading to repression or cleavage

L13 - Distinguish between properties that affect translation and those that affect mRNA stability

Incomplete complementary → translational repression

• the mRNA is not translated as effectively, so less protein is produced

Perfect complementary → mRNA cleavage

• the target mRNA is cleaved, reducing the amount of intact mRNA available for translation

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Translation is affected: the mRNA may still be present, but it is not used as effectively to make protein

mRNA stability is affected: the mRNA is broken down or becomes less available, so less intact mRNA remains to be translated

L13 - Summary slide

• Specific trans and cis acting factors, act together to regulate the rate of transcription initiation

• Epigenetic regulation affects DNA transcription

• Genes are alternatively spliced

• miRNAs repress gene expression post-transcriptionally

L14 - Explain how to isolate a gene that you want to clone

L14 - Describe the steps of inserting a gene into a plasmid and determining if you are successful

L14 - Summarize the steps of cloning

L14 - Design a vector that will allow you to express a human gene in bacteria