Aris Kaksis Riga University docent - Chemistry

Nucleoside PhosphorAmidate Monoesters Potential Pronucleotides RNA

Instructor: Dr. Natalia Tretyakova, Ph.D. «hyperlink "mailto:Trety001@umn.edu"»   -   6151
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Prof. e-mail: :ariska@latnet.lv

Required reading: Stryer 4th Ed. Ch. 34 p. 903-906

Synthesis: Take Home Message

1) DNA sequences are translated into RNA messages by RNA polymerases.

2) The initiation of RNA synthesis is controlled by specific DNA promoter sequences.

3) The synthesis of RNA is governed by initiation, elongation, and termination steps.

Flow of genetic information

DNA ||-------------> RNA -----------------------> Proteins ----------->>> Cellular action
Replication ||     transcription                  translation          -------->>>>>||||||||| 
           ||                    nucleare
    DNA nucleare <= Reverse transcription of telomeres

                                    Notable exception: retroviruses
RNA ||---> DNA ------------> RNA ------------------->   Proteins -------->>> Cellular action
Reverse || || transcription  transcription          translation       ------------>>>>>||||||||||| 
    DNA cytosolic      nucleare

Structural differences between DNA and RNA

DNA and RNA differ in several important ways:
1.) RNA is composed of Uracyl U and not Thymine T
2.) The sugar composition of RNA is composed of ribose and not de-oxy-ribose
3.) Sugar pucker on RNA is 3’ endo and RNA is usually single-stranded
(although it forms hairpins by folding over the same strand and makes the spliced tertiary 3° structure)

DNA	RNA
Thymine T 2'-de-oxy-ribose	Uracyl U D-ribose

RNA is a biopolymer consisting of four 4 nucleotide units

• usually RNA single stranded but can form loops and splice to form tertiary 3° structure:
                              --->C--->
                              U          G
                                         ¯
                              U         G
                                 \        /
                                  G=C <---
                                  A= U <--- Doublestranded
                                  C=G <--- Spliced tertiary
                                  C=G <--- 3° structure RNA
                                  G=C <---                   Loop
                                  C=G <---             formation
                                  C=G <---                Region
                                  G=C <---
5'---> U-C-C-C-A-G-/     \A-U-U-U --->3'                         There are Three Types of RNA

mRNA = Messenger RNA; an RNA copy of the DNA sequence (gene) used a template for
                                                                                                           protein synthesis

tRNA = Transfer RNA; a small 76 bp RNA that is attached to an amino acid AA which
                                                                                can be added to a growing peptide chain

rRNA = Ribosomal RNA; component of ribosomes with catalytic and structural function;
                                                                                     three types exist 23S, 16S, and 5S

Quantities of RNA in E. coli

Type           Relative amount      Mass # of Nucleotides
__________%_______________kDa____Number____

rRNA         80             23S          1.2•103   3700
                                    16S          0.6•103   1700
                                    5S            3.6•101     120

tRNA         15                              2.5•101       76

mRNA        5                                heterogeneous

RNA Polymerization Requirements

• Double-stranded DNA template strand
• GTP or ATP as the starting nucleotide (no primer)
• NTPs
• Mg2+

RNA Polymerase Structure

RNA polymerases from bacteria are very large (500kd) are composed of four subunits. a2bb's (holoenzyme)

Subunit      Number         Mass, kd    Function
a                   2                      37          Binds regulatory sequences
b                   1                    151          Forms phosphodiester bonds
b'                  1                    155          Binds DNA template
s                   1                      70          Recognizes promotor and initiates synthesis

RNA synthesis in E.Coli

1. Initiation: • search DNA to find promoter sites
                     • unwind a region of DNA to form replication bubble
                     • form the first 1st phosphodiester bond

2. Elongation • add NTPs to the growing chain following Watson-Crick base pairing rules

3. Termination • detect termination signals and pull RNA away from DNA duplex

Prokaryotic Promoter sequences

Promoter sequences for different genes                         ¯ Start site
Promoter for:          -35 region   Spacer         -10 region      Spacer    ¯ Transcribed
trp operon     G     TTGACA     N17       TTAACT        N7        A
tRNATyr         G    TTTACA      N16       TATGAT        N7        A
lP2                 G    TTGACA     N17       GATACT        N6        G
lac operon      C    TTTACA      N17       TATGTT        N6        A
rec A               C    TTGATA      N16       TATAAT        N7        A
lex A                G   TTCCAA      N17       TATACT        N6        A
T7A3               G   TTGACA      N17       TACGAT        N7        A
Consensus         TTGACA                  TATAAT
Initiation of RNA Polymerization

RNA Pol binding to promoter of DNA

A

RNA polymerase ¯ RNA Synthesis

RNA sequence is complementary to that of the transcribed DNA strand

Coding strand ||
5' ...ATGGCCTGGACTTCA... 3' Sense strand of       DNA
3' ...TACCGGACCTGAAGT...5'   Antisense strand of DNA
Non-coding (template) strand || Transcription of antisense strand
5' .. .A U G G C C U G G A C U U C A... 3'             mRNA
                                   || Translation of mRNA
           Met - Ala - Trp -   Thr - Ser -        Peptide

RNA vs DNA synthesis

Similarities:
          • require a DNA template
          • similar mechanism of nucleotide addition
          • Addition of nucleotides follows Watson-Crick base pairing rules

Differences:
          • RNA Pol does not require a primer
          • Unlike DNA Polymerases, RNA Polymerases do not have nuclease activity
          • RNA Pol is not able to proof read for mismatches
          • RNA synthesis is slower than DNA synthesis (50 nt/sec)

RNA Polymerase Holoenzyme

T7 RNA Polymerase

Termination of RNA Polymerization

                               --->C--->         Two 2 mechanisms possible :
                              U         G      1) stable hairpin formation
                              U          G       2) Rho (r) protein-mediated termination
                                 \       /
                                  G=C <---
                                  A= U <--- Doublestranded
                                  C=G <--- Spliced tertiary
                                  C=G <--- 3° structure RNA
                                  G=C <---                   Loop
                                  C=G <---             formation
                                  C=G <---                Region
                                  G=C <---
5'---> U-C-C-C-A-G-/     \A-U- U-U --->3'

r-independent termination

5'--->… UCCCAGCCCGCC U AAU RNA strand
                                                           G                                      unwinded antisence single strand DNA
                                                               A   GCCCG A A A A A A A A   C T
                                                                 G/ | | | | | |    |   |   |    |   |   | |             T
                                                                C \CGGGC U U U U U U U U -OH--->3'   G TT TT¬5'
                                                            T RNA polymerase => movement =>              |   | |   | |
DNA double strand                        C                                                                            C AA AA --->3'
3'<--- GGG TCG GGCGGAT TCA                                                                           A A
        | |   | |   | |   |   | |   | | | |   | | |                              T - T - T - T - T - T - T - G
5'--->CCCA GCCCGCCT A AGT G A G C G G G C unwinded sence single strand DNA

                                                               --->G--->         One of Two 2 mechanisms possible :
                                                              A          U      1) stable hairpin formation
                                                              A          G       2) Rho (r) protein-mediated

        A                                           termination
                                                                 C=G <---     Doublestranded
                                                                 G=C <---      Spliced tertiary
                                                                 C=G <---     3° structure RNA
                                                                 C=G <---       hairpin Loop
                                                                 C=G <---             formation
                                                                 G=C <---                Region
                                        5'--->C•C•C•A-/     \ U
                                                                            U       A A A A A A A C T
                                                                                U / |    |   |   |   |   | T
DNA double strand                                                 A \ U U U U U U -OH--->3'    G T TTT <---5'
3'<---GGG T CGG GC GGA T TCA CT CGCCCG                                               |   | |   | |
       | |   | |   | |   |   | |   | | |   |   | | |   | |   | | |   | | |       T T T T T T T T G AAC AA AA --->3'
5'--->CCCA GCC CG CCT AAGT GA GCG GGC unwinded sence single strand DNA

                                                               --->G--->
                                                              A           U
                                                              A          G
                                                                U      A
                                                                 C=G <---     Doublestranded
                                                                 G=C <---      Spliced tertiary
                                                                 C=G <---     3° structure RNA
                                                                 C=G <---          Stem Loop
                                                                 C=G <---             structure
                                                                 G=C <---
                                       5'--->C•C•C•A-/     \ U U U U U U U U U -OH--->3'
DNA double strand
3'<--- GGG T CGGGC GGAT TCA CTC GCCCG A A A A AA AA C TT G T TT T <---5'
          | |   | |   | |   | | |   | | | |   | | |   | | | | |   | | | |   |   |   |   | |   | |   |   | |   |   |   | |   |
5'--->CCC A GCCCG CCTA AG T GAGCGGGC T T T T T T T T G AA C A AAA --->3'

RNA Polymerization Reaction

5'

3'
3'

5'
¯ RNA Polymerase
5'

3'
3'

5'
s ¬¾¾¯ 17 bp unwound NTP's
¯ Coding Strand ¯
5'

PPP RNA-DNA 12 bp

Eukaryotic vs prokaryotic cell

Prokaryotes: • no membrane-bound nucleus   Eukaryotes: • DNA is in membrane-bound
• transcription and translation are coupled                                                              nucleus
                                                                                   • Transcription and translation are
                                                                                               separated in space and time
RNA splicing in eukaryotes

                                                                    gene

Chromosomal DNA
nuclear

Primary transcript hnRNA
RNA

¬RNA Splicing®

messenger mRNA
introns degrading to ® nucleotides                                                                                                           to cytosol

Eukaryotic RNA Polymerases

Type      Localization     RNA produced      -amanitin______actinomycin D

I             Nucleoli            rRNA                      Insensitive             strong

II            Nucleoplasm    pre-mRNA             Strongly inhibits   weak

III           Nucleoplasm    tRNA,5S rRNA     Weakly inhibits    weak

a-amanitin

RNA Pol II Inhibitor Amanita phalloides (the death cap)

Actinomycin D

Eukaryotic RNA Polymerase II

RNA Pol II is responsible for transcription to pre-mRNA

-        8-12 subunits

-        Two 2 large subunits ( 220kD and 140kD) responsible for synthesis

-        Regulated by phosphorylation of carboxyl-terminal domain (CTD)

-        some subunits are shared for RNA Pol I-III

Genes for Subunits of Yeast RNA Polymerase II

Gene	Protein Mass(kDa)	Deletion Mutant	*E.Coli* analog
RPB1 RPB2 RPB3 RPB4 RPB5 RPB6 RPB7 RPB8 RPB9 RPB10 RPB11 RPB12	190 140 35 25 25 18 19 17 14 8 14 7.7	nonviable nonviable nonviable nonviable conditional nonviable nonviable nonviable conditional nonviable nonviable nonviable	b b a - - - s

Core subunits

Shared subunits
Type II Eukaryotic Promoters

Consensus sequence:
-110 -40 -26 +1
5'

CAAT

GGGGCG

TATAAAA

CAAT Box GC Box TATA Box |--->| Start site
-------------------------------------> Promoter sequence---------------------------------->|| Coding sequence
Examples:

b-globin

SV40 barly

thymidine kinase

   histone H2B
    -120        -100         -80        -60         -40          -20         +1           +20
       |               |              |             |             |              |              |              |                |
        TATA Box -

GC Box -

(TATAAAA ) (GGGCGG)
CAAT Box -

Octamer -

(GGCCAATCT) ( ATTTGCAT)

Initiation of transcription in eukaryotes

• RNA Pol II can not initiate transcription by itself
• Transcription factors (TFII) are required
• The key initiation step is the recognition of TATA box by TBP

Enhancers
Looping of DNA

Formation of RNA Polymerase II pre-initiation complex

TFIID ®¯

IID contains TBP that binds TATA box
TFIIA ®¯

IIA stabilizes IID binding to promoter
TFIIB ®¯

IIB binds initiation sequence
RNA polymerase II + TFIIF ®¯

Pol II binds IIB
TFIIE + TFIIH ®¯

TATA Binding Protein

TBP Bends DNA

Hydrogen Bonds Necessary for TBP Binding

Enhancers can stimulate transcription from many nucleotides away
Enhancers
Looping of DNA

Figure 33-28 page 856

                           -200                          -100                                                    +1
                             |                                 |                                                          |
                                                ||<--------------------||                                                                                                             ||---------------------->
                             Late RNA ||                                                                 || Early RNA
SV40 ——————————————

—————————

||<-----------GC box ||------------>
|| || RNA
DHFR —————

————

—————

——————

————

||<----------GC box ||----------->
|| || RNA
Heat-shock gene —

———

—————

——

———
                             ||                                                                         TATA
                                                    ||<--------------Heat-shock element         Figure 33-29 page 857
Stryer: Biochemistry, Fourth Edition © 1995 by W.H.Freeman and Company.

1. Steroid hormone response elements (HRE) Estrogen, progesterone, glucocortecoids
2. P53 tumor suppressor gene.
Zinc Finger

"INDEPENDENCE OF METAL BINDING BETWEEN TANDEM CYS2HIS2 ZINC FINGER DOMAINS"
B.A. Krizek, L.E. Zawadzke, and J.M. Berg

                        Contents of file KRIZEK.KIN: 1ZAA.PDB
Kin.1 - Calpha trace of the Zif/268-DNA complex structure

1D66.PDB-Zn2+-Gal4
Leucine zipper 2ZTA.PDB

        Turn the sidechains back on, and notice that the interhelical contacts are made by sets of residues that look like wide rungs on a ladder (not, in fact, like teeth on a zipper). Every other rung contains a pair of orange leucines; alternate rungs contain pairs of gold valines, with one Met pair at the top and one Asn pair near the middle. In this kinemage, all of the Leu zipper sidechains are grouped and color-coded by their position in the seven-residue heptad repeat: 'd' (Leu) in orange; 'a' (Val) in gold, or in hotpink for Asn 16; 'e' and 'g', the contact-edge hydrophilics, in skyblue; and the outside positions 'b', 'c', and 'f' in cyan. Turn on the "heptad lbl" button to see labels a through g for one repeat.
        An end view of the Leu zipper supercoil shows the helix-helix contacts most clearly. Choose Reset2 under Graphics, "zoom, etc" under the Other menu, and zoom to enlarge the image. Turn off the "e, g" and the "b, c, f" buttons, to concentrate on the buried contact layers at positions a and d. For a detailed tour, set the zoom to 3.0, the zslab to 100, and move the slider to the top of the ztrans scrollbar; you should see just a little backbone and the sidechains of Met 2 at the end of each helix. Then hold down the mouse just above the arrow at the bottom of the ztrans scrollbar to move the structure fairly rapidly through the visible slab. A little more than one "rung", or layer, will be in view at a time. Notice how similar the geometry is for each rung of Leu 'd' sidechains; Val 'a' rungs are also similar to one another, but different in detail from Leu rungs. Leu Cbetas point toward each other and their Cgammas turn out; Val Cbetas point outward, while one of their Cgammas points inward to touch. Val is too short for optimal contact in a 'd' position, and Leu would not work in an 'a' position, because although its Cgamma could lie in the correct place its Cdeltas would then bump into the opposite helix.
        Turn on the "e, g" side chains and move the structure through the slab again by holding down the mouse just below the up arrow on the ztrans scrollbar. Notice how a given leucine contacts four surrounding sidechains on the opposite helix: its symmetry-mate Leu in 'd', the two adjacent 'a' residues, and the preceding 'g' residue. This kind of arrangement is called "knobs-into-holes" packing. It is usually not found in such a regular form in globular proteins, but was originally proposed by Francis Crick in 1953 to stabilize coiled coils.

RNA polymerase ||

                              Nascent RNA ||            || Cleavage signal               Figure 33-32, page 859 Stryer:
                              Nascent RNA ||<--- Cleavage by specific              Biochemistry, Fourth Edition 1995
                            Nascent RNA ||<---              endonuclease             by W.H.Freeman and Company
                                           ATP--->||<--- Addition of tail by
                                            PPi<---||<--- poly(A) polymerase
5' Cap

AA U AAA

AAAAAAAAAAAAAAAA(A)n
Polyadenylated mRNA precursor
RNA Synthesis: Take Home Message
1) DNA sequences are translated into RNA messages by RNA polymerases.
2) The initiation of RNA synthesis is controlled by specific DNA promoter sequences.
3) The synthesis of RNA is governed by initiation, elongation, and termination steps.