Transcription: DNARNA11/17 and 11/19

• RNA vs DNA how do their structures differ?• DNA Double helix and antiparallel H-bonds• What are codons and why are they important?• What are the steps to transcription in prokaryotes?• What are the steps to transcription in eukaryotes?• What are the RNApolymerase types?• What are transcription factors?• Introns and Exons: RNA processing• Where does tRNA and rRNA come from?

Suggestions for term paper drafts:Get another student or “four” to proof-read again just to double check your

changes.– If you like I can put papers in my box at office…you can put one in to be edited

and take one out to edit… do you want to voluntarily make this possible?Be sure each item in text relates specifically to your title, feel free to delete materials.

Over all length of final draft “text” is reduced to 3 page minimum to help you, although making it longer than 3 pages is fine.

Abstract vs. Conclusion: Make them differentthis is a tough one to do. – Brutal detail but short (no fluff) vs. Nice easy to read list/review of key points +

a possible sentence or two about what the future of this sort of research will be/Introduction: Tell the reader what topics you will discuss/teach and perhaps provide

a bit of background info if really needed/Try to have a minimum of two different references sources cited in each paragraph.

Try for at least 3 sentences or 4 lines of text in each paragraph.Write to remove “fluff” (it hurts to take things out that represent “neat” info) that is not

VERY specific to your paper “title”, if it does not relate to the title it probably should be removed

With regards to content in paper you need to look for repeated items/info and please remember: REPEAT DELETE or rephrase/reword

Bases of DNA and RNABase Choices for DNA: Adenine, Thymine, Cytosine and Guanine

Base Choices for RNA: Adenine, Uracil, Cytosine and Guanine

Base Pairing in DNA duplex: AT and GC

Base Coding from DNA to RNA: TRANSCRIPTION Transcription is performed by RNA Polymerase (RNAP)DNA bases transcribed into RNA basesAd-->Ur Td-->Ar Gd-->CrCd-->Gr

mRNA sequence (codons) are TRANSLATED into an amino acids linked by peptide bonds.

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Nucleoside consist of a ribose (3’ OH-sugar), base (A,T, G,C, or U) and a triphosphate. Link the phosphate to ribose with a phosphoester bond (cut off two phosphates) and your have RNA (R-OH) or DNA (R-H).

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Nucleotide remove PHOSPHATES left with a Nucleoside

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Hydrogen Bonds between two antiparallel nucleotide chains create the base pairs that stabilize your DNA (that’s why RNA is unstable)

TRANSLATION: The key to making a protein is to find the three ribonucleic acids on mRNA that make Methionine (met)

and to move 1 codon (triplet) and an additional amino acid/codon in the 3’ direction until a stop codon is reached.

Prokaryotic Transcription (RNA formation) is a four step cycle:• 1) Template Binding: template is recognized and bound by RNAP

at a special promotor sequence

• 2) Chain Initiation: a dinucleotide is formed from the template from ATP and an XTP– (X=Adenine, Guanine, Uracil, Cytosine plus ribose-5’ phosphate) (no

thymine in RNA: it is in DNA only)– After initiation the Sigma factor falls of RNAP

• 3) Chain Elongation: new bases are added to the nascent 3’ OH end of the mRNA.– Initiating factor gets bumped off after the first 10 bases

• 4) Chain Termination/Release: RNAP termination factor recognizes the termination sequence or a special RNA hairpin loop forms that lets RNAP fall off and mRNA is released– Many RNAs can be simultaneously produced from a single DNA strand.– Each strand of DNA can be used to make a unique mRNA depending

on the reading frame used to make initiate the RNA

Overview of Four Step Transcription Process:

Many mRNAs can be made at the same time from one DNA.The elongating chains below are mRNAs being transcribed.

RNAP must identify promotors -35 and -10 bases from where initiation begins (+1)! The -10 region is called a TATAAT or

Pribnow Box on the “5’3’) coding strand” [3’ATTCA5’ template strand]. This template strand becomes ‘5UAAGU3’ on mRNA.

The DNA duplex is unwound by RNAP to create access to the template strand for transcription into a complementary RNA

sequence. NTPs are ONLY added (elongation) to the RNA 3’ end

Termination occurs when a termination sequence of DNA is observed by the Rho factor of RNAP or when the RNA produced forms special complementary double stranded RNA sequences that pull RNAP off of the DNA. Normally RNA does NOT form a double stranded structure for large lengths/durations.

Eukaryotic Transcription (DNA RNA) is more complex, slower and more flexible than in Prokaryotes:

• Gross differences in prokaryotic and eukaryotic transcription.• 5 Eukaryotic RNA Polymerase subtypes• Core promoters on eukaryotic DNA• Transcription factors are required for RNAPolymerase-DNA

binding/transcription• Eukaryotic up-regulation and down-regulation of RNA production• Termination of RNA polymerase/transcription• Relative RNA content in eukaryotic cells• rRNA production by Polymerase I and processing• mRNA production by RNApolymerase II and processing• tRNA production by RNApolymerase III and processing• Removal of introns from pre-mRNA in the nucleus• Function of Spiceosomes

There are 6 major differences between eukaryotes and prokaryotes in-terms of how they approach transcription

• 1) E.T. have 5 different RNAPs, not 1– 3 in nucleus 1 in mitochondria 1 in chloroplast

• 2) E.T. promoters are much more variable– Variable with respect to identity and location

• 3) E.T. factors (TFs) bind DNA/initiate RNAP– Allows fine-tuning of RNA production

• 4) E.T. often have additional proteins that bind/modify TFs• 5) E.T. can cleave pre-RNA prior to final product formation• 6) E.T. can modify nucleotides of pre-RNA

Generally:Prokaryotes only make proteins when absolutely neededEukaryotes can make a bit more/less to fine tune production to

need

Relative to prokaryotes, eukaryotes have much greater flexibility in terms of their ability to modify protein production. This comes at the

cost of greater complexity and time needed to make a protein.

Eukaryotes have 5 different RNA polymerasesEach subtypes makes a specific type of RNA

1) RNAP I: makes rRNA in the nucleolus (darkest part)2) RNAP II: makes mRNA and snRNA in nucleoplasm3) RNAP III: makes rRNA and tRNA in nucleoplasm• RNAPolymerases are massive complexes: 500,000mw• Similar to Prokaryotic RNAPolymerases4) Mitochondria: has mRNAP5) Chloroplast: has cRNAP

How much of each type of RNA are contained in a cell? rRNA: about 75%

tRNA: about 15% mRNA: less than 10%

Transcription in Eukaryotes:

• A “core” promoter must be present near where RNA transcription is to begin!

• Many types of promoter exist

• Each RNAP type (I, II, III) looks for its unique promotor and synthesizes its unique RNA product type

• Promoter sequence on DNA may be upstream or downstream of initiation site – Upstream: before initiation site ( 5’ end of +1 on template DNA)– Downstream: after initiation site (3’ end of +1 on template DNA)

RNAP II makes mRNA! Several sequence patterns help make RNAP binding to mRNA possible: TATA-box, GC-box and the CAAT-box.

TATA Box is at -25 , not -10 as in prokaryotes• GC- and CAAT-boxes are variable located -40 to -100 bp

upstream of the +1 site: modify TATA-box/RNAP affinity:– They can be on the template OR coding DNA strands– They improve affinity of RNAP for TATA box– Classic eukaryotic promotors on mRNA:

Transcription Factor II core proteins let RNAPII bind DNA and begin transcription. Special TFs for RNAPI/RNAPIII also exist!• 1): TFIID- binds TATA DNA (often called a TBP)

• 2): A and B TFIIs bind/modify TFIID

• 3): RNAP II(TFIIF) can now bind at TATA box near +1

• 4): TFII-E binds and forms “pre-initiation complex”

• 5) Binding TFII-H permits DNA unwinding (helicase) and phosphorylation of RNAPII (activation)

• 6) Initiation occurs TFIIA/D released and transcription begins• Don’t forget that upstream histones and chromatins on DNA must

also be removed prior to DNA unwinding/transcription.• 7) Termination of mRNA transcription by RNAPII occurs when a

termination factor recognizes AAUAA in the RNA • Steps: #1D#2A/B#4RNAPII#5E/HA/D Release• Initiation complete and Transcription Begins!

“Transcriptional Regulation”: Eukaryotes have the unique ability to enhance/suppress transcription of a gene to better match needs for proteins that are non-constitutive in nature.

This is how steroid hormones work in cells to modify basal production so expensive protein production is fine tuned to need.

Enhancers/Silencers are generally 100s to 1000s of nucleotides away from the TATA box

What does the finished product look like when transcription is moving ahead?

Pre-mRNA is processed in several ways including: 1) Capping of the 5’ end with methyl-groups immediately after transcription2) Adding a poly A tail 3) Removal of “intron” sequences.

Introns located inside the pre-mRNA represent junk that must be properly removed by spliceosomes prior to departure from the nucleus. Spliceosomes recognize/cut consensus sequences OFF 5’ (GU) and 3’ end of (AG). Some mutations lengthen/shorten a protein by causing proteins to change their length and function (i.e. hemoglobin).

Spliceosomes are RNA-protein complexes that cut out unwanted pre-mRNA introns and link the remaining exons into a single mRNA.

Pre-mRNA is modified in many ways before exiting the nucleus and finding a ribosome for translated into a protein. Exons often become the functionally

distinct domains common to different proteins with similar/dissimilar functions.

RNAPI makes pre-rRNA. The 18S, 5.8S and 28S rRNAs are cut from a single larger pre rRNA. Methylations stabilize the rRNA structure/sequences

RNAPIII makes the tRNAs that carrying amino acids to the nascent proteins chains that produced from mRNA on a ribosome in the nucleus. tRNAs have a clover-leaf shape and hair-pin curves made possible by the RNA bases forming a DNA-like double strand. Notice that pre-tRNA nucleotides are removed, replaced and modified.