Genetics Lect 15

Gene

• segment of DNA used to make functional product (RNA or polypeptide)

• 1st Step: Transcription

Transcription

• copying of DNA sequence into RNA sequence

• proteins must recognize & act on DNA for it to occur

Gene Expression

overall process where info within a gene is used to produce a functional product which is used to (along w/ environmental factors) determine a trait

Regulatory Sequences

• site for binding of regulatory proteins

  • regulatory proteins: influence rate of transcription (variety of locations)

Promoter

• DNA sequences that “promote” gene expression by directing the exact location for initiation

• site for RNA polymerase binding

• signals beginning of transcription

  • located upstream from where transcription actually starts (near 5’ side)

Numbering System of Promoters

• Transcription Start Site (TSS): 1st nucleotide that acts as a template for transcription

• numbered in relation to start site

• Downstream (+): nucleotides located at right (3’ direction of coding strand)

• Upstream (-): nucleotide located to left (5’ direction of coding strand)

• Consensus Sequence: most common sequence (-35 and -10)

  • high levels of transcription

  • less if away from consensus sequence

Terminator

signals of of transcription

Ribosome-Binding Site

• site for ribosome binding to mRNA in bacteria

  • translation begins near this site in the mRNA

• In eukaryotes, the ribosome binds to a 7-methylguanosine cap in the mRNA, & the ribosome scans the RNA for a start codon

Codons

• codons: 3 nucleotide sequence that specify specific AA

• start codon: specifies 1st AA in polypeptide sequence

  • formylmethionine (bacteria) or methionine (eukaryotes)

• stop codon: end of polypeptide synthesis

  • polycistronic (bacteria): encodes 2+ polypeptides

Template Strand

DNA strand that is actually transcribed (used as template for complementary RNA sequence)

Coding Strand

• also called sense strand or nontemplate strand

• opposite strand of template strand

• identical to base sequence in RNA (except for Thymine)

Transcription Factors

recognize promoter & regulatory sequences to control transcription

What directs translation

ribosomal binding site & codons

3 Stages of Transcription

initiation, elongation, & termination (involves protein-DNA interactions)

Transcription Initiation

• promoter is a recognition site for transcription factors

• transcription factors enable RNA polymerase to bind to promoter

• After binding, DNA is denatured into bubble (Open Complex)

Transcription Elongation

• RNA polymerase slides along DNA in open complex to synthesize RNA

  • slides along template strand in 3’ to 5’ direction & RNA synthesized in 5’ to 3’ direction

  • pyrophosphate released

• open complex = 17 bases long & behind open complex, DNA rewind back into double helix

• 43 nucleotides/sec

Termination

• terminator is reached

• causes RNA-DNA hybrid of open complex separate

• RNA polymerase & RNA transcript to dissociate from DNA

2 Transcription Terminator Mechanisms (E. coli)

• rho-dependent termination

  • Requires a protein known as ρ (rho)

• rho-independent termination

  • Does not require ρ

How is the p-dependent termination facilitied

• a uracil-rich sequence located at the 3’ end of the RNA

• stem-loop structure upstream of the uracil-rich sequence

RNA Polymerase Holoenzyme structure

• complete, biologically active form of enzyme that is fully capable of initiating transcription at specific promoter site

• Core Enzyme: handles actual elongation of RNA but can’t recognize promoters on their own

  • 5 Subunits = α₂BB’w

• Sigma factor: regulatory subunit that binds to core enzyme to recognize & bind to promoter sequence

  • one subunit = σ

What does RNA Polymerase Holoenzyme do

• RNA polymerase holoenzyme binds loosely to the DNA & scans along the DNA, until it encounters a promoter

• When it does, sigma factor recognizes both the –35 and –10 sequences

• A region within the sigma factor that contains a helix-turn- helix structure is involved in a tighter binding to the DNA

RNA Polymerase & Complexes

• closed complex: binding of RNA polymerase to the promoter

• open complex: formed when the TATAAT box in the -10 sequence is unwound

  • A-T bonds are more easily separated

• A short RNA strand is made within the open complex

  • Sigma factor is released at this point

• marks the end of initiation

How many RNA polymerases are used in Eukaryotic transcription

RNA poly I, II, II

RNA Polymerase I

• Transcribes all rRNA genes (except for the 5S rRNA)

  • rRNA: combines w/ protein to form ribosome structure

RNA Polymerase II

• transcribes all protein-encoding (structural) genes (that is, it synthesizes all mRNAs)

• Transcribes some snRNA genes needed for splicing

  • short, non-coding RNA molecuels that mediate & catalyze pre-mRNA splicing

RNA Polymerase III

• Transcribes all tRNA genes (

  • translation, helps form polypeptide chain w/ AA

• Transcribes the 5S rRNA gene

• Transcribes microRNA gene

  • controls post-transactional gene expression

Eukaryotic Transcription Promoter

• have a core promoter & regulatory elements that influence the core promoter

  • core promoter (short): minimal portion of promoter required to properly intiate transcription

  • consists of TATA box (start area) & transcriptional start site (TATA not always present)

• Basal transcription: minimal, baseline level of gene expression w/o help of specific gene activators/repressiors

  • core promoter by itself produces a low level of transcription

Regulatory Elements

• Regulatory elements: short DNA sequences that affect the binding of RNA polymerase to the core promoter

  • Transcription factors (proteins) bind to these elements and influence the rate of transcription

• There are two types of regulatory elements

  • Enhancers: stimulate transcription

  • Silencer: Inhibit transcription

Cis-Acting Elements

DNA sequences that exert their effect only over a particular gene (TATA box, enhancers, silencers)

Trans-Acting Factors

regulatory proteins that bind to such DNA sequences (Cis-acting elements)

What 3 proteins are need for basal transcription to happen at the promoter

• RNA polymerase II

• Five different proteins called general transcription factors (GTFs)

• A protein complex called mediator

What are the two models for eukaryote termination

• Allosteric model: after transcribing the polyadenylation signal sequence, RNA polymerase II is destabilized & leaves DNA & termination occurs

• Torpedo model: an exonucelease binds to 5’ end of RNA that is being transcribed & degrades it in a 5’ to 3’ direction

Collinearity of Gene Expression

• a linear correspondence or ordered relationship at different levels of genetic organization

• Eukaryotes: protein-encoding genes aren’t always colinear w/ mRNA

• Ex: coding strand DNA sequence similar to mRNA sequence

pre-mRNA & Splicing

• beginning of transcription in the nucleus

• Introns (intervening sequence): non-coding region that are later removed or excised

• Exons: protein coding regions that are connected together or spliced

Processing of rRNA & tRNA Transcripts Modification

• DNA often made into larger, longer RNA molecules after being transcribed

• Transcript must be cut & processed into smaller, functional pieces

5’ Capping Transcript Modification

• modified guanine nucleotide (7-methylguanosine) is added to beginning (5’ end) of mRNA & is recognized by cap-bidning proteins

• Functions:

  • splicing of introns

  • helps mRNA leave nucleus & enter ribosome

  • helps ribosome recognize/attach to mRNA

3’ polyAtailing of mRNA transcripts

• Poly-A Tail: a long chain of adenine (A) nucelotides added to 3’ end of mRNA after gene is transcribed

• Functions:

  • protect mRNA from being broken down

  • inc. mRNA lifespan

  • help w/ translation

RNA Editing for Transcript

• base sequence of RNA changed after is has been transcribed

• Ex:

  • addition/deletion of particular base

  • conversion of base

  • Deamination: converts RNA nucleotides to new forms

Covalent Base Modification for Transcript

covalent modification of base within RNA

What are the 3 splicing mechanisms

• Group I intron splicing

• Group II intron splicing

• Spliceosome (pre-mRNA)

All three cases involve: Removal of the intron RNA & covalent linkage of the exon RNA fragments

Self-Splicing

• group I and II introns

• does not require the aid of enzymes

• Instead the RNA itself functions as its own ribozyme

  • RNA can fold into specific 3D shape that allows it to catalyze its own splicing/removal

• can occur in vitro without any proteins (outside cell)

• However, in vivo, maturase proteins often enhance the rate of splicing

Group 1 Splicing

• involves binding of a free guanosine to a site within the intron-leads to cleavage at 3’ end of exon 1

• Then bond between different nucleotide in the intron and the 5’ end of exon 2 is cleaved

• The 3’ end of exon 1 then forms a covalent bond with the 5’ end of exon 2

Group 2 Splicing

similar except that the 2’ OH group in adenosine begins catalytic process

Spliceosome

• pre-mRNA splicing requires the aid of a multicomponent structure known as the spliceosome

• It is composed of several subunits known as snRNPs (pronounced “snurps”)

• Each snRNP contains small nuclear RNA and a set of proteins

What are the functions of the spliceosome

• Bind to an intron sequence and precisely recognize the intron-exon boundaries

• Hold the pre-mRNA in the correct configuration

• Catalyze the chemical reactions that remove introns and covalently link exons

• Occurs via a metalloribozyme (a ribozyme that requires Mg 2+ ) in the U6 SnRNP

Alternative Splicing

• pre-mRNA with multiple introns can be spliced in different ways:

  • generate mature mRNAs with different combinations of exons

  • Two (or more) polypeptides can be derived from a single gene

  • organism to carry fewer genes in its genome, yet have a large diversity of polypeptide

Splicing Factors

• proteins that regulate alternative splicing

• Splicing Repressors: prevent splicing

• SPlicing Enhancers: facilitate splicing