Chap 7: DNA to protein

-- aminoacyl-tRNA activation --> 1 ATP
-- initiation --> 1 GTP
-- elongation step 1 --> 1 GTP for each aa added
-- elongation step 3 --> 1 GTP for each aa added
-- termination --> 1 GTP

- genes are regions of DNA that are transcribed into RNA
- mRNA allows the cell to separate information storage from information utilization
- transcription - copying in same “language”
- translation - goes from nucleic acid language to protein language

- many RNAs contain complex 3D shape
- Shape imp. for function
- folding driven by complementary base pairs
- single strand with double-stranded regions

--the flow of information in a cell
- mRNA separates
info storage from info utilization
- signal amplification of mRNAs
- rRNA and tRNAs
are nonspecific for polypeptide being made

DNA to RNA
- DNA template strand provides information for the synthesis of an RNA strand
- mRNA, tRNA, rRNA
- One DNA strand copied, thus a single strand of RNA

Notice that the base identity on the non-template strand (coding strand) is equivalent to the RNA sequence

Both DNA strands are not used to make RNA
- there is the template (noncoding) and conding strand
- only the template strand is used to make mRNA

RNA polymerase does not start at random sites on the DNA
- By convention all orientation is relative to the non-template strand
- Both strands considered part of gene
- Upstream (5' end) Downstream (3' end)

- Both strands can encode genes (but not in same spot on DNA)
- very unusual (eukaryotes) if both strands in one area encode for a gene ex. yeast genome

- Promoter sequence orientation determines which strand is the template.
- Polymerase always moves 3’ to 5’ along template strand

- DNA-dependent RNA polymerase is the copying enzyme
- one of the DNA strands is the template
- promoter -- DNA binding site
- transcription factors -- proteins that help RNAP recognize promoter
- moves 3’-->5’ on template
- RNAn + NPPP --> RNAn+1 + PPi

- A --> T in replication, but A --> U in transcription
- Unlike replication, new RNA strand does not remain H-bonded, but is displaced and the DNA helix reforms
- Also because RNA is copied from only a limited region of the DNA, RNA molecules are much shorter
- Transcription does not require a primer

- Several RNAP molecules on single DNA strands
- Nearly immediate release of RNA from template allows many RNA copies to be made from a gene in a short time

1. Ribosomal RNAs (rRNA)
2. Transfer RNAs (tRNA)
3. Messenger RNAs (mRNA)

3 distinct RNAP (prokaryotes have 1)
RNAP-I -- larger (28S, 18S, 5.8S) ribosomal subunits (this polymerase transcribes only one gene = the ribosomal RNA gene )
RNAP-II -- mRNAs and small nuclear RNAs
RNAP-III -- tRNAs and small (5S) ribosomal subunit
mRNA,tRNA,rRNA -- all made as larger pre-RNAs that are processed by “cut and paste” reactions;
snRNAs (small nuclear; 100-300 nt) -- with proteins help process mRNAs
snoRNAs (small nucleolar) -- process rRNAs
miRNAs (micro) -- regulate gene expression
- Transcription unit (sequence of nt that codes for a single RNA)
- all preRNAs are made as larger forms that are processed into smaller pieces

cells have millions of ribosomes
80% of RNA in cells is rRNA
rDNA are repeated 100’s of times
ribosomes consist of several rRNAs and dozens of proteins
half-life in days or weeks; gradually accumulate
~82 different proteins + 4 different rRNA molecules
- The genes for 18S, 5.8S, and 28S rRNA form a single transcription unit that is transcribed by RNA polymerase I to give a single large RNA (45S pre-rRNA)

Structure imp. for:
recognizing other molecules
structural support
catalyze aa covalent

- snoRNAs (small, nucleolar RNAs) help process pre-rRNA
- attach to rRNA while being transcribed

-- a ribosome is ~ 2/3 rRNA and 1/3 protein

- a ribosome- producing factory
- in interphase cells rDNA are clustered together in nucleolus
- most of nucleolus is ribosomal subunits
- in HeLa cells ~7,000 ribosomes are produced in the nucleolus/min
- GFP-ribosomal protein

plants and animals have ~50 different tRNAs
tDNA is repeated (~ 500 genes in humans)
repeats are clustered in genome
~ 80 nt long
transcribed using RNAP-III
half-life in days or weeks; gradually accumulate

- contains a continuous sequence of nt encoding a particular polypeptide
- significant noncoding segment (5’ and 3’ ends; regulatory functions)
- half-life varies; minutes to days
- Unlike ribosome RNA half life varies (bacteria half lives determined when they are needed)

-RNAP-II
- GTF -- General transcription factors
- promoter -- special sequence of DNA 24-32 nt “upstream” (5’) to transcription coding region

consensus sequence used by many genes
- key component of many promoters used by RNA polymerase II to from complete transcription initiation complex — order of assembly differ from one promoter to the next (usually); usually 30 nucleotides upstream from the start site. Once TFIID is bond to TATA box — other factors assemble — to from the initiation complex. Both bacteria and eukaryotic promoters are composed of several DNA sequences — to orient RNA polymerase for DNA template strand

- Through the promoters that help mRNA polymerase to know where to start copying.
- Rich TATATA (25 nucleotides areas of richness) tells mRNA polyermerase where to start copying — proteins will bind to it to tell the protein where to start binding
- Proteins recognize certain sequences

there are many GTFs in this complex; two imp. are:
-- TFIID (contains TBP)
-- TFIIH (contains a helicase and kinase)
Tail aka C-terminal domain (CTD)

A. most eukaryotic promoters contain a DNA sequence called the TATA box
B. The TATA box is recognized by a subunit of the general transcription TFIID called the TATA-binding protein (TBP); ** The DNA disruption produced b TBP is not shown
C. TFIID binding enables adjacent binding of TFIIB
D. The rest of the general transcription factors (as well as the RNA polymerase itself) — assemble at the promoter

- the C-terminal domain (CTD) of a RNAP-II subunit is phosphorylated by TFIIH
- phosphorylation releases polymerase from promoter site
- TFIID remains bound to TATA to initiate additional transcription
- Phosphorylation of the tail of RNA polymerase II allows RNA processing proteins to assemble there. Capping, polyadenylation, and splicing are all modification that occur as the RNA is being synthesized. The phosphates in this image is in addition to the ones required for transcription initiation.

modifies the 5’ end of the RNA transcript — the RNA cap includes an atypical nucleotide — the mRNA is simply the first nucleotide of the transcript. In eukaryotic cells, capping takes place after RNA polymerase II produced around 25 nucleotides of RNA before transcribing the whole gene
- increase the stability of mRNA molecule — and generally mark the RNA molecule as mRNA — they are also used by protein synthesis to make sure both ends of mRNA are present

hnRNA (heterogeneous nuclear RNA) is a pre-mRNA that is processed to mRNA
1. -- 5’ cap
2. -- 3’ polyadenylation (may occur during or after splicing)
3.--removal of introns
- big piece (hnRNA) trimmed to smaller pieces
- only 5% make it to the end (a lot is lost)
- all of this is done in the nucleus before going into the cytoplasm

- removal of a phosphate and addition of a methylated guanosine
- protects 5’ from exonucleases
- aids in transport from nucleus
- helps initiate translation
- 5’ cap is needed for starting translation once in the cytoplasm, recognized by proteins to help get into the cytoplasm

- endonuclease forms new 3’ end by recognizing AAUAAA sequence
- a polymerase adds 200-250 adenosines without template
- protects from premature degradation ** Going through 300 amines before getting to the source (eats things that do not matter)
- helps initiate translation