BIOL 304 UNIT 3 Lectures PDF

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Exam 3 - Monday April 3

- Note revised schedule - Exam 3 date remains unchanged - Exam 2 data should be posted on Friday March 17 - Schedule for painting White Hall is unknown and I will post the exam 2 answer key outside of my office (WHI 314A) and in the bulletin board next to WHI 126 - No office hours Thursday March 30; make an appointment for earlier in the week 1

Lecture and discussions on this topic do not align

3.1 03/15/17 Prokaryotic Transcription 19:509-548 -  Transcription produces a mRNA chain identical in sequence to the message / coding strand mRNA has U -  Several terms have been used to describe either the template or message / coding strand

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-  Published gene sequences = message strand from 5′ to 3′ -  5′ (left) to 3′ (right) -  Both strands may have ORFs -  RNA polymerase reads the template strand from 3′ to 5′ -  Arrow to right: top strand = message; 5′ to 3′ -  Arrow to left: bottom strand = message; 5′ to 3′ gene 1 gene 2 gene 1

5′ 3′

3′ 5′

gene 2

Image courtesy of A. Andres

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-  RNA synthesis is always in the 5′ to 3′ direction -  Template is 3′ to 5′ -  Ribonucleotides are added to the 3′ end; RNA has U and not T -  Both DNA strands can undergo transcription, but above rules apply

Introduction to Genetic Analysis, 11th

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Many prokaryotic genes are organized in operons -  Consists several genes under the control a single promoter -  Genes usually have related function RNAP binds to promoters -  Inducible operon -  Activated by an inducer protein -  Repressible operon -  Deactivated by a repressor protein

polycistronic mRNA – long mRNA with regions corresponding to different genes within an operon http://www.dnaftb.org/33/animation.html

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-  All genes have a promoter = binding site for RNA polymerase -  Upstream of transcriptional unit; promoter sequences not found in mRNA or cDNA -  Start point = 1st base that is transcribed into RNA (+1) -  Upstream sequences = before the start point (neg numbes) -  Downstream sequences = after start point (pos numbers)

Genes XI

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-  DNA strands separate -  Transcription bubble = 12-14 bp -  RNAP does not require a primer -  Adds a ribonucleotide triphosphate (ATP, CTP, GTP, UTP) to the 3′ end of the growing strand; RNAP looks for a 3′ - OH -  Catalyzes the formation of phosphodiester bonds -  Releases pyrophosphate; PPi Genes XI

-  As bubble moves = DNA duplex re-forms behind it, the mRNA exists in form of a single polynucleotide chain 7

Prokaryotic Transcription Overview

Initiation

Image from Discussion Manual

Elongation

Termination (Rho dependent or Rho independent)

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-  Prokaryotes have a single RNAP for mRNA, tRNA and rRNA -  Euk have 3 different RNAPs Pol I for larger rRNA; Pol II for most mRNAs; Pol III for tRNAs and smaller rRNAs Genes XI

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Bacterial RNA Polymerase Structure Complete enzyme = holoenzyme = 6 polypeptides; α2ββ′ωσ Divided into 2 parts: - Core enzyme (α2ββ′ω) -  Binding DNA and mRNA catalysis - Sigma (σ) factor -  Needed for promoter recognition

Genes XI

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Fig 19.7 uses the obsolete term eubacterial.

E. coli genes for the RNAP subunits -  Determine the function by mutational analysis -  Knockout each gene and assay the effect on transcription and shape of the holoenzyme -  α subunits help assemble the core and contact the promoter -  β and β′ are the catalytic subunits and form the main channel through which DNA passes, 2° channel for NTPs and the exit channel for the mRNA

Genes XI

Omega (ω) involved in enzyme assembly and regulation 11

-  Sigma (σ) factors control binding to DNA by recognizing specific sequences in promoters -  Core enzyme has a general affinity for DNA -  Electrostatic interactions btwn protein and DNA -  Core enzyme can synthesize DNA on a template in vitro, but

Genes XI

cannot recognize promoters -  Sigma factor ensures bacterial RNAP initiates transcription, and reduces binding to nonspecific sequences -  Sigma domains that interact with different promoter regions -  Bacteria have several sigma factors (Fig 19.15) for transcription of different gene under particular environmental conditions -  The initiation complex (Fig 19.13) requires the appropriate sigma factor

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Sigma factors -  Determine which promoters the RNAP core can bind -  A number of σ for special situations: stress, sporulation, N2 assimilation, chemotaxis -  σ70 is generic factor; replacement permits RNAP to recognize a different set of promoters

Genes XI

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Prokaryotic Promoter

Image from Discussion Manual

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Terms to Know -  Consensus sequence - the calculated order of most frequent residues found at each position in a sequence alignment -  Conserved sequence - similar or identical sequences which occur in DNA; these sequences occur across species.

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Consensus sequence illustration

Illustration also shows the 5′ untranslated region (5′ UTR) 5′ UTR is transcribed but not necessarily translated Important for ribosome binding; regulation of translation Introduction to Genetic Analysis, 11th

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Promoter sequences -  Promoter = DNA sequence/ 3D-structure -  75 bp in bacteria -  cis-acting site (on this side; same piece of DNA) -  non-coding DNA which regulate the transcription of nearby (200 bp) genes - Three locations important for this discussion -  Startpoint -  -10 -  -35

TATA box Pribnow +1 Genes XI

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-  (-10) sequence (starts anywhere from -5 to -9) consensus: TATAAT = T80A95T45A60A50T96 -  Subscript = the % occurrence of most frequently found base -  Conclude that increased frequency indicates importance for promoter recognition -  Also referred to as the Pribnow-Schaller box -  (-10) Engages the sigma and core; involved in both duplex and ss interactions -  (-35) sequence (centered at -35) consensus: TTGACA = T82T84G78A65C54A45 A = T; 2 H bonds -  (-35) Engages sigma Presume easier to separate the strands -  Distance (16-18 bp) separating the 2 regions is important in the orientation of RNAP with DNA -  Specific sequence of the intervening 16-18 bp is relatively unimportant 18

Start point is typically (>90%) a purine (A or G) Very common for the purine to be the central base in the sequence CAT Other elements of importance - Discriminator = immediately upstream of +1; binds sigma domain - Extended (-10) = bases between the -10 and -35 elements are in contact with a sigma domain - Upstream (20 bp of -35) promoter element (UP) -  AT rich and usually found in highly expressed promoters; has binding sites for the carboxy terminal domain (CTD) of α

Genes XI

Ext refers to the extended - 10 element

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Image from Discussion Manual

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-  All the component fit together in a very specific manner -  Shape of polypeptides complements the shape of the DNA; -10 and region 2 of sigma (Fig 19.23) -  Basic amino acids are attracted to negative charges of DNA -  Sigma is required for binding, but is released after the start of transcription Introduction to Genetic Analysis, 11th

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-  RNAP initially binds promoter region from -55 to +20 -  Fig 19.22 illustrates results from nuclease experiments that define points of contact -  Regions at -10 and -35 contain most of the points of contact

message strand template strand Genes XI

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RNAP is a molecular machine -  Unwinds and rewinds DNA -  Breaks and reforms H bonds -  Holds the separated strands and mRNA -  Incorporates complementary NTPs

Genes XI

-  Model for enzyme movement is based on its crystal structure -  Core enzyme provides a channel -  Lined with (+) charges -  jaws have a top and a bottom -  Clamp (top) bends DNA 90°

http://www.bio.miami.edu/dana/250/250SS14_8.html

-  Bridge (bottom) structure acts as a ratchet -  Rudder forces RNA chain out of complex -  Nucleotides assumed to enter from below

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Elongation

-  -  -  - 

Elongation is not at a steady pace Rate varies Determined by sequence RNAP can pause / arrest and even backtrack

-  RNAP unwinds DNA roughly 12-14 bp at a time -  Adds a nucleotide to the 3′ end of the growing strand -  Incorporates ribonucleotide triphosphates and catalyzes the formation of phosphodiester bonds -  Releases pyrophosphates 24 -  Stalled RNAP can restart by cleaving the mRNA at the 3′ end

Supercoiling is an important feature of transcription - Negatively supercoiling = twisting against the helical conformation (left handed twisting); underwinds or straightens the DNA; releases tension; melts the DNA - Positive supercoiling = right-handed, double-helical conformation of DNA is twisted even tighter (right-handed twisting); helix begins to knot -  RNAP unwinds and rewinds -  Generates (+) supercoils ahead and (-) supercoils behind -  For each helical turn +1 is generated ahead and -1 turn behind

Gyrase introduces negative supercoils

Genes XI

-  Gyrase introduces (-) supercoils via ds breaks -  Topoisomerase I removes (-) supercoils via ss breaks

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Prokaryotic Termination - Transcription continues beyond the end of the gene -  Creates a 3′ untranslated region (3′ UTR) - RNA pol elongates until a terminator is reached; two models in prokaryotes 1  Intrinsic terminators; do not need cellular factors; determined by mRNA sequence or hairpin formation 2  Rho (ρ) dependent terminators - Termination = nucleotides no longer incorporated, chain released, RNAP dissociates from DNA template - Note: bacterial rRNA genes and some phages have anti-terminators; produces a long RNA molecule that is processed into appropriate lengths; topic covered in other classes 26

Intrinsic Terminators -  ½ of all E. coli genes -  RNA pol stalls when it transcribes lots of Us -  DNA has A = mRNA has U -  Stalling may cause mRNA to dissociate from the template -  Stalling does not always lead to dissociation -  Probably depends on length of sequence Intrinsic terminators also have palindromic regions can form G-C hairpins -  mRNA comes loose from template -  Up and down stream sequences are important -  The RNA being produced by RNAP plays an important role in termination

Genes XI

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Introduction to Genetic Analysis, 11th

Image from Discussion Manual

Genes XI

Rho-dependent terminators -  Need rho (ρ) factor -  Rho binds at rut (rho utalization) site of mRNA -  C rich and G-poor region -  No 2° structure; no hairpins -  Tracks along RNA to move into RNAP environment -  When catches up (often compared to a race); unwinds DNA/RNA hybrid, causes RNAP to fall off -  rut sites are not restricted to the 5′ of the mRNA; can be found near the intrinsic termination site

Genes XI

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-  Rho protein = hexameric protein -  Binds RNA -  ATP-binding site; ATPase domain -  Helicase domains -  Nucleic acid passes through the doughnut hole (Fig 19.33) Genes XI

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Transcription and translation are coupled in prokaryotes RNA polymerase

-  No separation in prokaryotes Transcription & translation occur

mRNA

in same space

chromosome

-  Ribosomes can associate with transcript while it is still being made -  Prokaryotic mRNA has ribosome binding sites; Shine Dalgarno

ribosome

polypeptide RNA polymerase

mRNA

sequences chromosome 31

Prokaryotic mRNA must be translated and there are regions of mRNA that important for this process. UTR = untranslated region 32

Comparison of Prokaryotic Replication and Transcription Replication

Transcription

Template

DNA

DNA

Product

DNA

RNA

2

1

DNA polymerase

RNA polymerase

dNTPs

NTPs (ATP, GTP, UTP, CTP)

Enzyme binding site

Origin of Replication

Promoter

Start site

Origin of Replication

+1

Replicon

Transcriptional unit

Primer needed

yes

No

Proofreading

yes

No (exceptions)

Reading direction

3’-5’

3’-5’

Direction of synthesis

5’-3’

5’-3’

Increases over time

Stays the same

bidirectional

unidirectional

# of strands synthesized Major enzyme Type of nucleotide used

Region of synthesis

Bubble size Directionality of syn.

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Eukaryotic Transcription vs Prokaryotic Transcription Prokaryotes

Eukaryotes

Promoter

Usually before the start point

Around the start point

Enzyme binding to promoter

RNA polymerase

Transcription factors

Name of TAATAT consensus

Pribnow box

TATA box

Location of TAATAT consensus

-10

-25

Number of RNA polymerases

1

3

Proteins needed for initiation

Sigma Factors

General transcription factors

Does RNA polymerase bind directly to the promoter?

Yes

No

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Bacterial mRNA

Genes XI

-  mRNAs contain 5′ un-translated region (UTR) and 3′ UTR -  5′ UTR is also known as the leader sequence or leader RNA -  Starts at transcription start (+1) and ends 1 nt before the start codon (AUG) -  3′ UTR immediately follows the translation stop codon(s) and has regulatory regions important for stability, translatability, localization

Genetics: A Conceptual Approach, 5th

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The 30S (small) subunit binds to Shine-Dalgarno element - AG rich sequence on 5′ UTR mRNA complementary to a conserved sequence close to the 3′ end of 16S rRNA -  SD is 8-10 bases upstream of the start codons: AUG, GUG and UUG -  SD assists in aligning the ribosome with the start codon

-  5′...AGGAGG-N~10-AUG...3′ -  SD is complementary to the 3′ end of 16S rRNA -  3′...UCCUCC...5′ 5

-  -  -  -  - 

mRNA is fragile when compared with DNA RNAs are ss and are more susceptible to ribonucleases RNases are either endonucleases vs exonucleases Endoribonucleases have an internal recognition sites Exoribonucleases degrade specific ends; 5’- 3’ vs 3’- 5’ exoribonucleases

Genes XI

-  In euks, mRNA has a 5’ G-cap and poly(A) tail block ribonucleases -  Bound ribosomes, RNA binding proteins, stem loop structures protect mRNA by blocking ribonucleases 6

-  mRNAs are not designed to last forever -  Degradation mRNA is a mechanism to control gene expression -  Prok RNAs are generally short lived (~3 min) -  Prok have stem loop structures that prevent degradation -  Prok lack the 5’ G-cap and 3’ poly(A) modifications of eukaryotes Page 115 Discussion Manual

-  In proks, transcription and translation are simultaneous -  Polysomes produces a large amount of polypeptide -  Ribosomes protect mRNA from degradation 7

mRNA stability: Prok vs Euk Prokaryotes mRNA relative stability

Lower (1-3 min) *90 min had been reported

page 115 discussion manual Eukaryotes Higher (up to 24 hours)

mRNA lifespan Minutes Structure helping achieve stability Message is degraded when

Location of transcription and translation General degradation mechanism

Stem loop

Ribosomes are not packed densely

Minutes to days 5’cap (guanine) and poly(A) tail As messsage ages, adenylases remove A’ s to an oligo(A) approx 10’s

Same place - cytoplasm

Different places – nucleus and cytoplasm

mRNA degraded by degradosome

6 pathways: topic for future lecture

-  mRNAs are not designed to last forever -  Degradation mRNA is a mechanism to control gene expression

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Bacterial mRNA degradation occurs in several repeated steps via degradosome -  Terminal PPi cleaved from 5′ end -  Endonuclease (RNaseE) cuts 5′ end to generate 3′-OH -  Free 3′-OH chewed up by 3′ to 5′ exonuclease: PNPase = Poly-Nucleotide Phosphorylase -  As ribosomes move, more degradation -  Several other components -  Enolase: function not clear; enzyme from glycolysis

Genes XI

-  Helicase: ATP dependent removal of stem loop structures 9

Degradosome = degrades mRNA - RNaseE, PNPase, enolase, helicase (RhlB)

prok mRNAs differ greatly in their half-life -  20 seconds to 90 min (shorter than average for euks) -  Factors affecting half-life: -  PNPase are unable to process ds regions = stem loop structures -  RNase E is also impeded by stem loop -  High ribosome density due to efficient translation also blocks attachment of degradosome http://www.biochemsoctrans.org/bst/035/0502/bst0350502a01.gif

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Operon = operational unit -  Many prokaryotic and viral genes are organized in operons -  Present in euks, but not very common -  Consists several genes under the control a single promoter -  Polycistronic message -  1 mRNA; each cistron usually has a SD, start and stop codon -  Genes usually have related function -  Requires a regulatory gene(s) somewhere else on the chromosome and not necessarily nearby -  Regulatory gene is constitutively expressed Nice review of eukaryotic operons at http://www.ncbi.nlm.nih.gov/pubmed/15642184

Constitutive genes

Terminology

-  Actively expressed all the time -  Housekeeping genes -  Required to keep the cell alive -  Glycolysis, citric acid cycle, ETC, active transport, plus others -  Regulated via allosteric activators & inhibitors Inducible genes -  Expressed only when their proteins are needed by the cell -  Cell growth, DNA synthesis, mitosis, meiosis, metabolism of a unique carbon source, stress response, plus many others

Terminology

Positive regulation -  Gene expression = transcription

-  Activator protein(s) recognizes and binds to a regulatory sequence near or overlapping the promoter

Activator protein = necessary for or increases transcription

Terminology Negative regulation -  Repressor protein recognizes and binds to a sequence near to or overlaps the promoter

Repressor protein = prevents or stops transcription

Terminology

cis-actings element same side = same molecule -  Not necessary immediately upstream of the operon genes -  Constitutively expressed; regulatory gene lacI is cis -acting trans-acting proteins -  Bind to a short sequence near or part of the promoter -  Activators & repressors are trans-acting action from a different molecule

-  Inducible operon

Terminology

-  Normal state is off -  Turned on by an inducer -  Inducer present during a particular environmental condition or stimulus -  Repressible operon -  Normal state is on -  Turned off by a repressor -  Repressor present during an environmental condition / stimulus; negative feedback regulation -  The main advantage of operons is that a single switch can either turn on or off a cluster of function...