1. Lecture:11
Dr.Q.M.I.Haq

2. Transcription
The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNA.
The whole genome of DNA needs to be replicated, but only small portion of genome is transcribed in response to the development requirement, physiological need and environmental changes.
DNA regions that can be transcribed into RNA are called structural genes.

3. Example from DNA Replication to RNA Transcription
TAC GAT ACT GGA CCA CAT CAG Old DNA coding strand (sense strand)
ATG CTA TGA CCT GGT GTA GTC New DNA template strand (antisense strand)
UAC GAU ACU GGA CCA CAU CAG mRNA
coding strand
template strand
template strand
coding strand
The template strand is the strand from which the RNA is actually transcribed. It is also termed as antisense strand.
The coding strand is the strand whose base sequence specifies the amino acid sequence of the encoded protein. Therefore, it is also called as sense strand.

4. RNA Polymerase
The enzyme responsible for the RNA synthesis is DNA-dependent RNA polymerase.
The prokaryotic RNA polymerase is a multiple-subunit protein of ~480kD.
Eukaryotic systems have three kinds of RNA polymerases, each of which is a multiple-subunit protein and responsible for transcription of different RNAs.

5. Recognition of Origins
Each transcriptable region is called operon.
One operon includes several structural genes and upstream regulatory sequences (or regulatory regions).
The promoter is the DNA sequence that RNA-pol can bind. It is the key point for the transcription control.

6. Consensus sequence Prokaryotic promoter
The -35 region of TTGACA sequence is the recognition site and the binding site of RNA-pol.
The -10 region of TATAAT is the region at which a stable complex of DNA and RNA-pol is formed.

7. Transcription Process
General concepts
Three phases: initiation, elongation, and termination.
The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process.
The eukaryotic RNA-pol requires co-factors to bind to the DNA template together in the transcription process.
Transcription of Prokaryotes
Initiation phase: RNA-pol recognizes the promoter and starts the transcription.
Elongation phase: the RNA strand is continuously growing.
Termination phase: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template.

8. a. Initiation
RNA-pol recognizes the TTGACA region, and slides to the TATAAT region, then opens the DNA duplex.
The unwound region is about 171 bp.
No primer is needed for RNA synthesis.

9.          b. Elongation
The  subunit releases and the remaining subunits of holoenzyme (known as core enzyme) slides on the DNA template toward the 3 end.      
 subunit releases   

10. RNA-pol, DNA segment of ~40nt and the nascent RNA form a complex called the transcription bubble.
The 3 segment of the nascent RNA hybridizes with the DNA template, and its 5 end extends out the transcription bubble as the synthesis is processing.
Transcription bubble

11. -dependent termination
c. Termination
The RNA-pol stops moving on the DNA template. The RNA transcript falls off from the transcription complex.
The termination occurs in either  -dependent or  -independent manner.
-dependent termination
The termination function of  factor

12. Post-Transcriptional
Modification
The nascent RNA, also known as primary transcript, needs to be modified to become functional tRNAs, rRNAs, and mRNAs.
The modification is critical to eukaryotic systems.

13. Modification of hnRNA
Primary transcripts of mRNA are called as heteronuclear RNA (hnRNA).
hnRNA are larger than matured mRNA by many folds.
Modification includes
Capping at the 5- end
Tailing at the 3- end
mRNA splicing
RNA edition
Exons are the coding sequences that appear on split genes and primary transcripts, and will be expressed to matured mRNA.
Introns are the non-coding sequences that are transcripted into primary mRNAs, and will be cleaved out in the later splicing process.

14. Differences between replication and transcription
Similarity between replication and transcription
Both processes use DNA as the template.
Phosphodiester bonds are formed in both cases.
Both synthesis directions are from 5´ to 3´.
Differences between replication and transcription
replication
transcription
template
double strands
single strand
substrate
dNTP
NTP
primer
yes no
Enzyme
DNA polymerase
RNA polymerase
product
dsDNA
ssRNA
base pair
A-T, G-C
A-U, T-A, G-C

15. Protein Synthesis (Translation)
The information encoded in the DNA is transferred to messenger RNA and then decoded by the ribosome to produce proteins.
RNA Translation
It is the process of translating RNA message (from mRNA) into proteins with the help of transfer RNA (tRNA).
This uses an mRNA sequence as a template to guide the synthesis of a chain of amino acids that forms proteins.
Translation occurs in the cytoplasm where the ribosomes are located.

16. RNA Translation
Initiation - A ribosome attaches to the mRNA and starts to code at the FMet codon (START codon: AUG, GUG or UUG).
2. Elongation - tRNA brings the corresponding amino acid to each codon as the ribosome moves down the mRNA strand.
3. Termination - Reading of the final mRNA codon (STOP codon: UAA, UAG, UGA), which ends the synthesis of the peptide chain and releases it.
Specifically, the code defines a mapping between tri-nucleotide sequences called codons and amino acids.
Every triplet of nucleotides in a nucleic acid sequence specifies a single amino acid.

17. A series of three adjacent bases in an mRNA molecule codes for a specific amino acid—called a codon.
A triplet of nucleotides in tRNA that is complementary to the codon in mRNA—called an anticodon.
Each tRNA codes for a different amino acid.
Genetic code

18. Proteins are composed of amino acids – there are 20 different amino acids
Different proteins are made by combining these 20 amino acids in different combinations
Three tRNA binding sites:
A site = amino-acyl tRNA binding site
P site = peptidyl-tRNA binding site
E site = exit site
The association of the large and small subunits creates the structural features on the ribosome that are essential for protein synthesis 18

19. Transfer RNA (tRNA) carries amino acids from the cytoplasm to the ribosome.
mRNA carrying the DNA instructions and tRNA carrying amino acids meet in the ribosomes.

20. Amino acids are joined together to make a protein.
Polypeptide = Protein

21. Protein synthesis DNA unwinds
mRNA copy is made of one of the DNA strands.
mRNA copy moves out of nucleus into cytoplasm.
tRNA molecules are activated as their complementary amino acids are attached to them.
mRNA copy attaches to the small subunit of the ribosomes in cytoplasm. 6 of the bases in the mRNA are exposed in the ribosome.
A tRNA bonds complementarily with the mRNA via its anticodon.
A second tRNA bonds with the next three bases of the mRNA, the amino acid joins onto the amino acid of the first tRNA via a peptide bond.
The ribosome moves along. The first tRNA leaves the ribosome.
A third tRNA brings a third amino acid
Eventually a stop codon is reached on the mRNA. The newly synthesised polypeptide leaves the ribosome.

22. 5’-ATGCCTAGGTACCTATGA-3’ 3’-TACGGATCCATGGATACT-5’
DNA
Transcription
5’-AUGCCUAGGUACCUAUGA-3’
mRNA
decoded as
5’-AUG CCU AGG UAC CUA UGA-3’
Translation
N-MET-PRO-ARG-TYR-LEU-C
Protein

23. In prokaryotes a single transcript can give rise to multiple proteins.
Polycystronic mRNAs
Monocystronic mRNAs

24. Mutation What could happen when things go wrong?
A mutation is a permanent change in the DNA sequence of a gene. Mutations in a gene's DNA sequence can alter the amino acid sequence of the protein encoded by the gene.
Most mutations are neutral or harmless. These are called silent mutations. However, when mutations affect the composition of a gene they can cause illness or even death.
Thesunwashotbuttheoldmandidnotgethishat.
The sun was hot but the old man did not get his hat.
This sentence represents a gene. Each letter corresponds to a nucleotide base, and each word represents a codon. What if you shifted the three-letter "reading frame”?

25. Mutation Thesunwashotbuttheoldmandidnotgethishat.Or
Th esu nwa sho tbu tth eol dma ndi dno tge thi sha t.
Mutations are usually due to mistakes that are made when cells copy their DNA before dividing and that are not detected by the cell’s repair mechanisms.
Other mutations may be caused by outside mutagenic agents such as ultraviolet rays and other forms of radiation, chemicals, toxins, and drugs.

27. Mutations that happen in body cells may cause an illness like cancer.
Breast cancer cell
BRCA1 gene
Angelman Syndrome
Down Syndrome
Only mutations that happen in germ cells (egg or sperm cells) are passed from parent to offspring.
In this case, the mutation will be in every cell of the offspring.